Nucleic acid aptamer-based diagnostic methods with novel techniques for signal enhancement

ABSTRACT

The present invention concerns methods for the detection of target molecules in a sample including several steps of signal amplification allowing the detection of a very low number of target molecules in the tested sample. The detection assay is based on the use of a universal probe which enables the signal amplification. The specific recognition of the target molecule is achieved by using a specific binding agent, preferably an aptamer. The invention further concerns kits and methods for the diagnosis of pathological conditions.

FIELD OF THE INVENTION

This invention relates to diagnostic methods and kits. In particular,the present invention provides novel techniques for enhanced detectionand quantitation of target molecules based on a universal detectionprobe.

BACKGROUND OF THE INVENTION

There is an increasing need for accurate, fast and low cost assays inthe fields of in-vitro diagnostics (IVD) and especially in the field ofPoint of Care (POC) diagnostics. Nucleic Acid aptamers have beenconsidered in recent years for use in diagnostic applications.

Aptamers are oligonucleic acid molecules which are obtained by in vitroselection methods such as Synthetic Evolution of Ligands by ExponentialEnrichment (SELEX).

Aptamers have a specific binding to a target molecule such as a proteinwith high affinities in the nanomolar and subnanomolar range. Forexample, aptamers against vascular endothelial growth factor andkeratinocyte growth factor show affinities of 100 pM and of 1 pM,respectively (L R Paborsky et al., “The Single-Stranded DNAAptamer-Binding Site of Human Thrombin” J. of Biol. Chem. 268 (1993):808-811). In addition, aptamers can bind small molecules like ATP (MSassanfar and J W Szostak, “An RNA Motif That Binds ATP” Nature 364(1993): 550-553).

In recent years, aptamers have been studied as therapeutic tools bothfor treatment and for prevention, with the first aptamer-based drugrecently approved by the U.S. FDA in treatment for age-related maculardegeneration (AMD).

Several aptamer-based biodetection approaches have been reported inwhich aptamers were labeled with molecules such as redox probes,fluorescent dyes, or nanocrystals as an integral part of signaltransduction (Mir M, and Katakis I. Aptamers as elements ofbioelectronic devices. Mol. Biosyst. 3 (2007): 620-622; Levy, M; Cater,S F; Ellington, A D. Quantum-dot aptamer beacons for the detection ofproteins. Chembiochem. 6 (2005):2163-2166). In addition, researchershave recently taken advantage of PCR to amplify DNA aptamers forsensitive detection of protein targets.

In addition, in the past several years there was a growing use ofaptamers as chemical antibodies (William James. Aptamers. InEncyclopedia of Analytical Chemistry. R. A. Meyers (2000) 4848-4871 ÓJohn Wiley & Sons Ltd; Pai S S, Ellington A D, Using RNA aptamers andthe proximity ligation assay for the detection of cell surface antigens.Methods Mol. Biol. 504 (2009): 385-398).

U.S. Pat. No. 6,458,543 describes a nucleic acid ligand “biochip”,consisting of a solid support to which one or more specific nucleic acidligand is attached in a spatially defined manner, enabling the specificbinding to a target molecule, if present, in a test mixture.

WO 2007/117444 describes methods for diagnosing and staging diseases bydetecting and/or measuring proteins associated with certain clinicalconditions using a plurality of aptamers that recognize oligopeptideepitopes on a target protein.

WO 01/79562 describes a novel aptamer based two-site binding sandwichassay, employing nucleic acid ligands as capture and/or reportermolecules.

Mir and Katakis (Mir M, and Katakis I. Aptamers as elements ofbioelectronic devices. Mol. Biosyst. 3 (2007):620-622) describe anaptamer based-assay having sensitivity of detection in the range of10⁶-10¹² molecules.

SUMMARY OF THE INVENTION

The present invention is based on the development of a detection assaywhich includes several steps of signal amplification allowing thedetection of a very low number of target molecules in a tested sample.The detection assay is based on the use of a universal probe whichenables signal amplification. The specific recognition of the targetmolecule is achieved by using a specific binding agent, preferably butnot limited to an aptamer. Using the methods disclosed herein theuniversal probe is attached to the specific biding agent and largelyamplifies the detection signal.

Accordingly, by a first of its aspects, the present invention provides amethod for the detection of a target molecule in a sample comprising:

a. obtaining at least one aptamer capable of binding to said targetmolecule, wherein said at least one aptamer is bound to a matrix;

b. incubating said at least one aptamer which is bound to the matrixwith the sample under conditions allowing the binding of the aptamer tothe target molecule; thereby forming a matrix-aptamer-target moleculecomplex;

c. contacting the matrix-aptamer-target molecule complex formed in step(b) with a polymer associated with a member of an affinity couplewherein said polymer further comprising a reactive group; therebyforming a matrix-aptamer-target molecule-polymer complex; and

d. contacting said matrix-aptamer-target molecule-polymer complex with acomplementary member of said member of an affinity couple, wherein saidcomplementary member is associated with a detectable moiety,

wherein the amount of said detectable moiety is indicative of thepresence of said target molecule in the sample.

In another aspect, the present invention provides a method for thedetection of a target molecule in a sample comprising:

a. obtaining at least one aptamer capable of binding to said targetmolecule, wherein said at least one aptamer is bound to a matrix;

b. incubating said at least one aptamer which is bound to the matrixwith the sample under conditions allowing the binding of the aptamer tothe target molecule; thereby forming a matrix-aptamer-target moleculecomplex;

c. contacting the matrix-aptamer-target molecule complex formed in step(b) with a nucleic acid molecule comprising a reactive group and furthercomprising a polymerase promoter sequence, thereby forming amatrix-aptamer-target molecule-nucleic acid molecule complex;

d. adding a DNA or RNA polymerase enzyme and nucleotides associated witha member of an affinity couple under suitable conditions to affect DNAor RNA polymerization, thereby obtaining DNA or RNA molecules associatedwith a member of an affinity couple, and

e. contacting said DNA or RNA molecules associated with a member of anaffinity couple with a complementary member of said member of anaffinity couple associated with a detectable moiety;

wherein the amount of said detectable moiety is indicative of thepresence of said target molecule in the sample.

In another aspect, the present invention provides a method for thedetection of a target molecule in a sample comprising:

a. obtaining at least one first binding agent capable of binding to saidtarget molecule, wherein said first binding agent is bound to a matrix;

b. incubating said at least one first binding agent which is bound tothe matrix with the sample under conditions allowing the binding of thebinding agent to the target molecule; thereby forming a matrix-bindingagent-target molecule complex;

c. contacting the matrix-binding agent-target molecule complex formed instep (b) with a second binding agent-polymer complex, wherein saidsecond binding agent-polymer complex is obtained by either

-   -   i. obtaining at least one biotinylated second binding agent;    -   ii. incubating said at least one biotinylated second binding        agent with streptavidin thereby a biotinylated second binding        agent streptavidin (b-binding agent-SA) complex is formed; and    -   iii. incubating said b-binding agent-SA complex formed in        step (ii) with a polymer associated with a member of an affinity        couple wherein said polymer further having a reactive group        thereby forming a second binding agent-polymer complex; or    -   iv. obtaining at least one second binding agent, wherein said at        least one second binding agent comprises a reactive group; and    -   v. incubating said at least one second binding agent comprising        a reactive group with a polymer associated with a member of an        affinity couple wherein said polymer further having a reactive        group thereby forming a second binding agent-polymer complex;

d. contacting the matrix-binding agent-target molecule complex formed instep (b) with the second binding agent-polymer complex formed in step(c), under conditions allowing the binding of the second binding agentto the target molecule, thereby forming a target molecule-polymercomplex;

e. contacting said target molecule-polymer complex formed in step (d)with a complementary member of said member of an affinity coupleassociated with a detectable moiety,

wherein the amount of said detectable moiety is indicative of thepresence of said target molecule in the sample.

In another aspect, the present invention provides a method for thedetection of a target molecule in a sample comprising:

a. obtaining at least one first binding agent capable of binding to saidtarget molecule, wherein said first binding agent is bound to a matrix;

b. incubating said at least one first binding agent which is bound tothe matrix with the sample under conditions allowing the binding of thebinding agent to the target molecule; thereby forming a matrix-bindingagent-target molecule complex;

c. contacting the matrix-binding agent-target molecule complex formed instep (b) with a second binding agent-nucleic acid complex, wherein saidsecond binding agent-nucleic acid complex is obtained by either

-   -   i. obtaining at least one biotinylated second binding agent;    -   ii. incubating said at least one biotinylated second binding        agent with streptavidin thereby a biotinylated second binding        agent streptavidin (b-binding agent-SA) complex is formed; and    -   iii. incubating said b-binding agent-SA complex formed in        step (ii) with a nucleic acid having an active group and further        comprising a polymerase promoter sequence thereby forming second        binding agent—nucleic acid complex; or    -   iv. obtaining at least one second binding agent, wherein said at        least one second binding agent comprises a reactive group; and    -   v. incubating said at least one second binding agent, comprising        a reactive group with a nucleic acid having a reactive group and        further comprising a polymerase promoter sequence thereby        forming a second binding agent-nucleic acid complex;

d. contacting the matrix-binding agent-target molecule complex formed instep (b) with the second binding agent-nucleic acid complex formed instep (c), under conditions allowing the binding of the second bindingagent to the target molecule;

e. adding a DNA or RNA polymerase enzyme and nucleotides associated witha member of an affinity couple under suitable conditions to affect DNAor RNA polymerization, thereby obtaining DNA or RNA molecules associatedwith a member of an affinity couple, and

f. contacting said DNA or RNA molecules associated with a member of anaffinity couple with a complementary member of said member of anaffinity couple associated with a detectable moiety;

wherein the amount of said detectable moiety is indicative of thepresence of said target molecule in the sample.

In another aspect, the present invention provides a method for thediagnosis of a pathological condition in a subject comprising using adetection method in accordance with the invention as described above,wherein said target molecule is a target molecule associated with thepathological condition and wherein the amount of said detectable moietyis indicative of the presence of a pathological condition in thesubject.

In another aspect, the present invention provides a method formonitoring the efficiency of a therapeutic regimen in a subjectsuffering from a pathological condition comprising using a detectionmethod in accordance with the invention as described above, wherein saidtarget molecule is an antigen associated with the pathological conditionand wherein the amount of said detectable moiety is indicative of thelevel of the pathological condition and thereby of the efficiency of thetherapeutic regimen in the subject.

In another aspect, the present invention provides kits for affecting thedetection methods of the invention.

Accordingly, in one embodiment the present invention provides a kitcomprising:

(a) at least one aptamer; and

(b) a polymer wherein the polymer is associated with a member of anaffinity couple and wherein said polymer is further associated with areactive group.

In another embodiment the present invention provides a kit comprising:

(a) at least one aptamer; and

(b) a nucleic acid molecule comprising a reactive group and furthercomprising a polymerase promoter sequence.

In yet another embodiment the present invention provides a kitcomprising:

(a) a first binding agent; and

(b) a second binding agent—polymer complex, wherein the polymer isassociated with a member of an affinity couple and wherein said polymeris further associated with a reactive group.

In yet another embodiment the present invention provides a kitcomprising:

(a) a first binding agent; and

(b) a second binding agent-nucleic acid complex, wherein the nucleicacid in said complex comprises a reactive group and further comprises apolymerase promoter sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIGS. 1A and 1B show schematic representations of modified matrix (1A)aptamers (denoted by arrows) immobilized on pre-coated magnetic beads(1B) aptamers (denoted by arrows) immobilized on pre-coated magneticbeads, which are additionally coated with cross-linkers (denoted asblack dots).

FIGS. 2A and 2B are schematic representations of a biotinylated polymer,which is incubated with the antigen-aptamer-matrix complex (2A) Ageneral formula of the biotinylated polymer, which consists of nrepeating units, carries a reactive group and binds to a succinimidylester (SE) group (2B) A specific example of the polymer being abiotinylated double strand-DNA carrying a succinimidyl group(“b-ds-DNA-SE”).

FIG. 3 shows a schematic representation of the complex used in the “dualaptamer assay”, which is formed by the streptavidin (SA)-aptamer complexand the b-ds-DNA-SE.

FIGS. 4A and 4B show schematic representation of the amplificationmethods according to the invention (4A) enzyme-labeled amplificationobtained by the addition of streptavidin covalently coupled to eitherhorseradish peroxidase (HRP) enzyme or alkaline phosphatase (AP), whichbinds to the biotinilayed polymer, followed by the addition of therelated substrate. (4B) Additional amplification obtained by convertingthe ds-DNA, by using the T7 or SP6 promoter sequences located on theds-DNA and the addition of biotinylated NTP in the T7/SP6 RNA polymerasereaction, into biotinylated RNA, which may then be transferred to anavidin labeled matrix for detection.

FIGS. 5A to 5D show a schematic representation of the branched DNA assay(5A) the ss-DNA-SE complex formed by incubating the thiol labeled ss-DNAwith s-SMPB or s-SMCC, which reacts with thiol residual groups on thess-DNA (5B) the aptamer-SA-ss-DNA complex formed by binding of the SEgroup from the ss-DNA to the primary NH₂ groups of the SA (5C) Apre-prepared ss-DNA sequence directed branch unit comprising AP and HRP(5D) the branch unit (according to the selected directed ss-DNAsequences) attached to the ss-DNA

FIG. 6 shows a schematic illustration of the “single aptamer assay”.

FIG. 7 shows a schematic illustration of the “dual aptamer assay”.

FIG. 8 shows a plasmid carrying the Hepatitis B scrambled sequence.

FIG. 9 is a graph showing the detection of TNF-α by (A) unmodified TNF-αantibody (B) TNF-α antibody attached to a b-DNA-SE group (C) TNF-αantibody attached to a b-DNA-SE group and using an additional step of T7RNA amplification.

FIGS. 10A and 10B are bar graphs showing the detection of recombinantPDGF-BB in PBS and in FCS and of native PDGF-BB in human serum usingPDGF-BT-2 aptamer attached to the matrix (10A) signal detection usingbDNA-SE (10B) Employing a dual aptamer assay by using PDGF1 aptamerattached to SA-b-DNA for detection.

FIGS. 11A and 11B are bar graphs showing the detection of PDGF-BB inserum using PDGF-2 aptamer attached to the matrix, by differentamplification methods (11A) b-DNA-SE (11B) a T7 RNA polymerase reaction.(1:10 of Ag).

FIGS. 12A and 12B are bar graphs showing the detection of PDGF-BB withthe branched DNA technology using (12A) ss-DNA-SE (12B) ss-DNA IgG.

FIGS. 13A-13C are graphs showing the detection sensitivity of PDGF-BBantigen using the PDGF2 aptamer and b-DNA-SE by fluorometric substrateor chromogenic substrate (13A) AttoPhos® Alkaline phosphates fluorescentsubstrate system and a Tecan fluorometer (13B) AttoPhos® Alkalinephosphates fluorescent substrate system and a Qubit® Fluorometer (handsize) (13C) pNPP Alkaline Phosphatase chromogenic substrate.

FIGS. 14A and 14B are bar graphs showing the specificity of detectingrecombinant CMV-gB antigen using (14A) the CMV-gB1 aptamer (14B)scrambled aptamer.

FIG. 15 is a bar graph showing the specificity of detecting cultured CMV(1.4×10e4 PFU) spiked into either PBS/BSA buffer or into human serumusing the CMV-gB1 aptamer.

FIGS. 16A and 16B are bar graphs showing the detection of Listeria withaptamers (16A) specificity of the Ap03 and Ap08 aptamers to differentListeria strains (16B) concentration dependent studies of the Ap03 andAp08 aptamers and of both aptamers on the same matrix in Listeria strainLS4.

FIG. 17 is a bar graph showing the detection of Listeria bacteria (10e⁵PFU) by a dual aptamer assay using Ap03 and Ap08 aptamers.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention provides a novel method for the detection of lowlevels of target molecules in a sample using a universal detection probewhich enables amplification of the detection signal.

The method of the present invention is highly sensitive and can be usedto detect as low as 10³ antigen copies or target molecules in a testedsample.

In addition, the method of the invention is accurate and providesresults within a short time frame. It also involves low cost tests whichin general do not involve the use of sophisticated equipment.

The following first two aspects of the invention are generally referredto herein as a “single binding agent assay” specifically “single aptamerassay”. The “single binding agent assay” relates to the use of theuniversal detection probe in conjunction with a nucleic acid aptamer.

Thus, in accordance with the first aspect, the present inventionprovides a method for the detection of a target molecule in a samplecomprising:

a. obtaining at least one aptamer capable of binding to said targetmolecule, wherein said at least one aptamer is bound to a matrix;

b. incubating said at least one aptamer which is bound to the matrixwith the sample under conditions allowing the binding of the aptamer tothe target molecule; thereby forming a matrix-aptamer-target moleculecomplex;

c. contacting the matrix-aptamer-target molecule complex formed in step(b) with a polymer associated with a member of an affinity couplewherein said polymer further comprising a reactive group; therebyforming a matrix-aptamer-target molecule-polymer complex; and

d. contacting said matrix-aptamer-target molecule-polymer complex with acomplementary member of said member of an affinity couple, wherein saidcomplementary member is associated with a detectable moiety,

wherein the amount of said detectable moiety is indicative, of thepresence of said target molecule in the sample.

In accordance with the second aspect, the present invention provides amethod for the detection of a target molecule in a sample comprising:

a. obtaining at least one aptamer capable of binding to said targetmolecule, wherein said at least one aptamer is bound to a matrix;

b. incubating said at least one aptamer which is bound to the matrixwith the sample under conditions allowing the binding of the aptamer tothe target molecule; thereby forming a matrix-aptamer-target moleculecomplex;

c. contacting the matrix-aptamer-target molecule complex formed in step(b) with a nucleic acid molecule comprising a reactive group and furthercomprising a polymerase promoter sequence, thereby forming amatrix-aptamer-target molecule-nucleic acid molecule complex;

d. adding a DNA or RNA polymerase enzyme and nucleotides associated witha member of an affinity couple under suitable conditions to affect DNAor RNA polymerization, thereby obtaining DNA or RNA molecules associatedwith a member of an affinity couple, and

e. contacting said DNA or RNA molecules associated with a member of anaffinity couple with a complementary member of said member of anaffinity couple associated with a detectable moiety;

wherein the amount of said detectable moiety is indicative of thepresence of said target molecule in the sample.

The following two aspects of the invention are generally referred hereinas “two binding agents assay”. The “two binding agents assay” relates tothe use of the universal detection probe in conjunction with at leasttwo binding agents.

In accordance with the third aspect, the present invention provides amethod for the detection of a target molecule in a sample comprising:

a. obtaining at least one first binding agent capable of binding to saidtarget molecule, wherein said first binding agent is bound to a matrix;

b. incubating said at least one first binding agent which is bound tothe matrix with the sample under conditions allowing the binding of thebinding agent to the target molecule; thereby forming a matrix-bindingagent-target molecule complex;

c. contacting the matrix-binding agent-target molecule complex formed instep (b) with a second binding agent-polymer complex, wherein saidsecond binding agent-polymer complex is obtained by either

-   -   i. obtaining at least one biotinylated second binding agent;    -   ii. incubating said at least one biotinylated second binding        agent with streptavidin thereby a biotinylated second binding        agent streptavidin (b-binding agent-SA) complex is formed; and    -   iii. incubating said b-binding agent-SA complex formed in        step (ii) with a polymer associated with a member of an affinity        couple wherein said polymer further having a reactive group        thereby forming a second binding agent-polymer complex; or    -   iv. obtaining at least one second binding agent, wherein said at        least one second binding agent comprises a reactive group; and    -   v. incubating said at least one second binding agent comprising        a reactive group with a polymer associated with a member of an        affinity couple wherein said polymer further having a reactive        group thereby forming a second binding agent-polymer complex;

d. contacting the matrix-binding agent-target molecule complex formed instep (b) with the second binding agent-polymer complex formed in step(c), under conditions allowing the binding of the second binding agentto the target molecule, thereby obtaining a target molecule-polymercomplex;

e. contacting said target molecule-polymer complex formed in step (d)with a complementary member of said member of an affinity coupleassociated with a detectable moiety,

wherein the amount of said detectable moiety is indicative of thepresence of said target molecule in the sample.

In accordance with the fourth aspect, the present invention provides amethod for the detection of a target molecule in a sample comprising:

a. obtaining at least one first binding agent capable of binding to saidtarget molecule, wherein said first binding agent is bound to a matrix;

b. incubating said at least one first binding agent which is bound tothe matrix with the sample under conditions allowing the binding of thebinding agent to the target molecule; thereby forming a matrix-bindingagent-target molecule complex;

c. contacting the matrix-binding agent-target molecule complex formed instep (b) with a second binding agent-nucleic acid complex, wherein saidsecond binding agent-nucleic acid complex is obtained by either

-   -   i. obtaining at least one biotinylated second binding agent;    -   ii. incubating said at least one biotinylated second binding        agent with streptavidin thereby a biotinylated second binding        agent streptavidin (b-binding agent-SA) complex is formed; and    -   iii. incubating said b-binding agent-SA complex formed in        step (ii) with a nucleic acid having an active group and further        comprising a polymerase promoter sequence thereby forming second        binding agent-nucleic acid complex; or    -   iv. obtaining at least one second binding agent, wherein said at        least one second binding agent comprises a reactive group; and    -   v. incubating said at least one second binding agent, comprising        a reactive group with a nucleic acid having a reactive group and        further comprising a polymerase promoter sequence thereby        forming a second binding agent-nucleic acid complex;

d. contacting the matrix-binding agent-target molecule complex formed instep (b) with the second binding agent-nucleic acid complex formed instep (c), under conditions allowing the binding of the second bindingagent to the target molecule;

e. adding a DNA or RNA polymerase enzyme and nucleotides associated witha member of an affinity couple under suitable conditions to affect DNAor RNA polymerization, thereby obtaining DNA or RNA molecules associatedwith a member of an affinity couple, and

f. contacting said DNA or RNA molecules associated with a member of anaffinity couple with a complementary member of said member of anaffinity couple associated with a detectable moiety;

wherein the amount of said detectable moiety is indicative of thepresence of said target molecule in the sample.

The “sample” according to the present invention may be any sampleincluding, but not limited to, biological samples obtained from subjects(including humans and animals as detailed below), samples obtained fromthe environment for example soil samples, water samples, agriculturesamples (including plant and crop samples), or food samples.

In one embodiment, said sample is a liquid sample.

The term “subject” in accordance with the invention includes but is notlimited to a human, an animal, in particular, a primate, a householdanimal or an animal used in agriculture.

Furthermore, the term subject encompasses healthy subjects, subjectssuffering from various diseases, subjects receiving various treatments,as well as deceased subjects (e.g. for forensic analysis).

In some embodiments, the biological sample may be a bodily fluid, atissue, a tissue biopsy, a skin swab, an isolated cell population or acell preparation.

In certain embodiments the cell in the population of cells or cellpreparation is selected from an animal cell, a viral cell, a bacterialcell and a fungal cell.

In some specific embodiments, the population of cells comprises cancercells. In another embodiment the population of cells is an in vitrocultured cell population.

In some embodiments, the biological sample may be a bodily fluidselected from the group consisting of blood, serum, plasma, urine,cerebrospinal fluid, amniotic fluid, tear fluid, nasal wash, mucus,saliva, sputum, broncheoalveolar fluid, throat wash, vaginal fluid andsemen.

Samples according to the invention may be samples obtained from theenvironment for example soil samples or water samples. Water sample maybe obtained for example but not limited to from drinking water, sewage,sea water, lakes, and rivers. The method disclosed in the presentinvention may be applied for home use, municipal use, or governmentaluse.

Agriculture samples may also be used, for example plant samples and cropsamples. Plant samples refer to any plant or pare thereof being forexample seeds, fruit, or leaves and include but are not limited to fieldcrops or greenhouse-grown plants. The invention also encompasses plantsamples obtained from wild plants (i.e. plants which are not grown bymen).

Food samples may be obtained for example from fresh food, cooled food orfrozen food.

The term “target molecule” as used herein denotes a molecule which maybe found in a tested sample and which is capable of binding to a bindingagent.

The term “binding agent” as used herein refers to any molecule capableof specifically binding to the target molecule for example an aptamer,an antibody, a receptor ligand or a molecular imprinted polymer.

In one embodiment, the target molecule is an antigen.

As used herein the term “antigen” refers to a target molecule capable ofbinding to a binding agent. In accordance with some embodiments, theantigen is a soluble antigen (also termed herein a “circulatingantigen”), a cell-surface antigen, or an antigen associated with amicelle, a liposome or a particle.

In some embodiments, the antigen may be a protein, a polypeptide, apeptide, a ganglioside, a lipid, a phospholipid, a carbohydrate, a smallmolecule or a nucleic acid.

Non limiting examples a soluble antigen in accordance with the inventionare soluble cancer markers, inflammation-associated markers, hormones,cytokines, drugs, and soluble molecules derived from a virus, a bacteriaor a fungus for example, toxins or allergens.

In some embodiments, the antigen is a viral antigen. In the context ofthe invention the term “viral antigen” is to be understood as a proteinor fragment thereof encoded by the viral genome.

In some other embodiments, the antigen is a cancer (or tumor) marker. Ingeneral, a tumor marker may be found in the body fluids such as in bloodor urine, or in body tissues. Tumor markers may be expressed or overexpressed in cancer and are generally indicative of a particular diseaseprocess.

Non limiting examples of a cell surface antigen in accordance with theinvention are a receptor, a cell surface marker, a viral antigen, or areceptor ligand.

The method of the invention may have therapeutic uses for example it maybe used for the detection of various pathological conditions or may beused for monitoring the disease stage of a subject or its response totherapy.

Thus, in accordance with the fifth aspect, the present inventionprovides a method for the diagnosis of a pathological condition in asubject comprising using the detection methods of the invention asdisclosed above, wherein said target molecule is a target moleculeassociated with the pathological condition and wherein the amount ofsaid detectable moiety is indicative of the presence of a pathologicalcondition in the subject.

In addition, in accordance with the sixth aspect, the present inventionprovides a method for monitoring the efficiency of a therapeutic regimenin a subject suffering from a pathological condition comprising usingthe detection methods of the invention as disclosed above, wherein saidtarget molecule is an antigen associated with the pathological conditionand wherein the amount of said detectable moiety is indicative of thelevel of the pathological condition and thereby of the efficiency of thetherapeutic regimen in the subject.

The “pathological condition” according to the present invention may beselected from but not limited to cancer, inflammation, blood coagulationdisorders, and autoimmunity. Accordingly, the method of the inventionmay be used in the detection of known cancer markers, markers ofinflammation, such as Procalcitonin which is a known marker for sepsis,peptides such as penicillin-binding protein 2 (PBP2), kinesin pindleprotein (KSP), toxins and alergens.

The method of the invention may be employed in the detection of a viralinfection. Non limiting examples of viral infections are Hepatitis Bvirus (HBV), hepatitis C virus (HCV), Cytomegalovirus (CMV) (for examplefor the detection of CMV in transplanted patients and pregnant women),Human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), HERPESvirus, Polio virus and influenza virus (both Human and Avian).

The method of the invention may also be employed in the detection of abacterial infection. Non limiting examples of bacterial causinginfections are Listeria, Staphylococcus Aureus, Methicillin resistanceStaphylococcus Aureus (MRSA), Corynebacterium Diphtheriae (causingDiphtheria), E. coli, Group B streptococcus (GBS), Group Astreptococcus, Mycobacterium Tuberculosis (causing Tuberculosis (TB),Salmonella, Vibrio Cholerae, Campylobacter, Brucellosis, NeisseriaMeningitidis (causing meningococcus), Streptococcus pneumonia andCandida.

For example, the estimated number of MRSA tests, is about 40 million peryear, of which 15 millions are gene based tests. Rapid and appropriateantimicrobial therapy, including the administration of vancomycin, iscritical for effective treatment. However, conventional methods foridentifying MRSA, such as disc susceptibility testing, are not alwaysreliable since phenotypic expression of methicillin resistance is knownto be heterogeneous. As nucleic acid techniques such as PCR fordetecting the mecA gene (Zeeshan M, et al. comparison of differentphenotypic methods of detection of methicillin resistance instaphylococcus aureus with the molecular detection of mec-a gene. J.Coll. Physicians. Surg. Pak. 17 (2007):666-70) are expensive andtechnically demanding, simple and more inexpensive techniques arerequired for routine use.

In some embodiments, the pathological condition is cancer. Cancer isinterchangeably used with the terms malignancy, tumor and is referred toherein as a class of diseases in which a group of cells displayuncontrolled growth and invasion that may destroy adjacent tissues, andsometimes leads to metastasis (spreading to other locations in thebody). Cancer may be a solid cancer or a non-solid cancer and may beclassified as carcinoma, sarcome, lymphoma, leukemia, germ cell tumor,or blastoma.

In some further embodiments, the pathological condition is an autoimmunedisease. As appreciated in the art, autoimmune diseases arise from anoveractive immune response of the body against substances and tissuesnormally present in the body. Non limiting examples of autoimmunedisease are Multiple sclerosis, Arthritis Autoimmune hepatitis, Crohn'sdisease, Diabetes mellitus, type 1, Inflammatory bowel disease, Multiplesclerosis, Psoriasis, Rheumatoid arthritis, Wegener's granulomatosis.

Using the detection methods of the present invention the level of targetmolecules indicative of the pathological state may be determined.Therefore, the measurement of the levels of these target molecules canserve to diagnose the pathological condition, to monitor diseaseprogression and to monitor efficacy of a therapeutic regiment, i.e.monitor the response of the subject to treatment.

According to some aspects, the method of the invention is based on theuse of binding agents. In some embodiments, the binding agents may eachbe independently an aptamer, an antibody, a receptor ligand or amolecular imprinted polymer.

In some embodiments, the first binding agent is an aptamer.

In some embodiments, the second binding agent is an aptamer.

In some embodiments, at least one of the binding agents is a nucleicacid aptamers.

As used herein the term “complex” denotes an entity comprising more thanone molecule which is bound or is in association with at least one othermolecule, for example by a chemical association. Hence the term“matrix-aptamer-target molecule complex” relates to an associationbetween the matrix, aptamer and the target molecule. The term“matrix-aptamer-target molecule-polymer complex” relates to anassociation between the matrix, aptamer, target molecule and thepolymer. The term “matrix-aptamer-target molecule-nucleic acid moleculecomplex” relates to an association between the matrix, aptamer, targetmolecule and the nucleic acid. The term “matrix-binding agent-targetmolecule complex” relates to an association between the matrix, bindingagent and the target molecule. The term “second binding agent-polymercomplex” relates to an association between the second binding agent andthe polymer. The term “biotinylated second binding agent streptavidin(or b-binding agent-SA) complex” relates to an association betweenbiotin, a second binding agent and streptavidin. The term “targetmolecule-polymer complex” relates to an association between amatrix-binding agent-target molecule complex and a second bindingagent-polymer complex.

As used herein, the term “aptamers” or “specific aptamers” denotessingle-stranded nucleic acid (DNA or RNA) molecules which specificallyrecognizes and binds to a target molecule.

The aptamers according to the invention may fold into a defined tertiarystructure and can bind a specific target molecule with highspecificities and affinities (William James. Aptamers. In Encyclopediaof Analytical Chemistry. R. A. Meyers (2000) 4848-4871 Ó John Wiley &Sons Ltd; Pai S S, Ellington A D, Using RNA aptamers and the proximityligation assay for the detection of cell surface antigens. Methods Mol.Biol. 504 (2009): 385-398).

Aptamers are usually obtained by selection from a large random sequencelibrary, using methods well known in the art, such as SELEX and/orMolinex (William James. Aptamers. In Encyclopedia of AnalyticalChemistry. R. A. Meyers (2000) 4848-4871O John Wiley & Sons Ltd;Tombelli S, et al. Aptamers-based assays for diagnostics, environmentaland food analysis. Biomol Eng. 24 (2007):191-200).

According to the present invention and as appreciated in the art, therecognition between the aptamer and the antigen is specific and may bedetected by the appearance of a detectable signal by using acolorimetric sensor or a fluorimetric/lumination sensor.

The aptamers as used according to some aspects of the invention may bebiotinylated.

The aptamers may optionally include a chemically reactive group at the3′ and/or 5′ termini. The term reactive group is used herein to denoteany functional group comprising a group of atoms which is found in amolecule and is involved in chemical reactions.

Some non-limiting examples for a reactive group include primary amines(NH₂), thiol (SH), carboxy group (COOH), phosphates (PO4), Tosyl, and aphoto-reactive group.

In some embodiments, the aptamer as used herein may optionally comprisea spacer between the nucleic acid sequence and the reactive group. Thespacer may be an alkyl chain such as (CH2)_(6/12), namely comprising sixto twelve carbon atoms.

According to the present invention, the aptamers are bound to a matrix.The matrix according to the invention may be for example any supportstructure or a surface. Non limiting examples include beads, e.g.magnetic beads, agarose beads, sephadex beads, glass beads, flatsurfaces such as a culture plate, a well in a plate, a tube surface,quantum dots, resins, plastic paper, nitrocellulose membranes, particle,microsphere and Molecular imprinted Polymers (MIP) (Michael J. et al.The rational development of molecularly imprinted polymer-based sensorsfor protein detection, Chem. Soc. Rev., 40 (2011): 1547-1571).

The magnetic beads may be selected from but not limited toTosylactivated beads or Streptavidin coated “MyOne” and “M280”Dyna-Beads (BD, Invitrogen).

While working with magnetic beads, all related procedures are performedas directed by the manufacturer. All washings are performed by employinga Magnetic separator. FIG. 1 provides an example of magnetic beadssuitable for use in the present invention.

The matrix is initially pre-blocked in order to reduce or eliminate nonspecific binding by any suitable blocking agent known in the art. Nonlimiting examples of coating materials are protein, acryl amide,synthetic polymer and polysaccharides.

In some embodiments, coating may be for example by covalently binding ofblocking proteins such as bovine serum albumin (BSA), streptavidin,avidin, extravidin or any modification thereof.

In some further embodiments, coating may be by any low affinity bindingprotein or polymer.

In certain embodiments the matrix may be activated for example bypre-coating with a compound selected from a group consisting ofpolylysine, epoxy, tosyl, carboxylic acid, carboxylated polyvinylalcohol and photoreactive crosslinkers. Examples of photoreactivecrosslinkers include but are not limited to simple aryl azides,fluorinated aryl azides or benzophenone derivatives.

In some embodiments, an activated matrix includes molecularly imprintedpolymers (MIP), antibody associated, or ligand associated matrix.

As used herein the term “activated matrix” refers to a matrix which canbe covalently bound to a first binding agent, for example an aptamer ora suitably modified aptamer. Said activated matrix is obtained bycoating the matrix with a suitable material, for example as listedabove.

The first binding agent, for example the aptamers may be attached to thepre coated matrix, for example by covalently binding to the coatingprotein.

For example, in an embodiment whereby a streptavidin coated matrix isused, the first binding agent is biotinylated. For example biotinylatedaptamers may be preferably used.

According to this embodiment, the aptamers according to the differentaspects of the invention are biotinylated and the matrix is pre-coatedwith streptavidin, the aptamer is bound to the matrix via streptavidinbiotin binding, thereby forming an aptamer-matrix complex.

In an embodiment whereby a protein-coated matrix is used (for example, aBSA-coated matrix), primary NH₂ or SH labeled aptamers may be preferablyused by employing homobifunctional crosslinkers or heterobifunctionalcross linkers.

As used herein the term “crosslinkers” refers to crosslinking reagentswhich contain two or more reactive ends capable of chemically attachingto specific functional groups (primary amines, sulfhydryls, etc.) onproteins or other molecules.

According to some embodiments, the first binding agent of the presentinvention for example the aptamer, comprises reactive groups and thematrix is pre-coated with a protein, the first binding agent is bound tothe matrix by adding a cross linking agent, thereby forming a firstbinding agent-matrix complex. In some specific embodiments, anaptamer-matrix complex is formed.

Homobifunctional crosslinkers are reagents that have the same type ofreactive group at either end. Amine crosslinkers (namely bind aminereactive groups) may be selected for example from glutaraldehyde,bis(imidoesters) or bis(succinimidylesters) (also known as NHS esters).

According to some specific embodiments, homobifunctional crosslinkerssuch as but not limited to dimethyl pimelimidate (DMP) or Glutaraldehydecan bind to primary NH₂ groups on the BSA surface and to NH₂ groups theaptamers. Sulfhydryl crosslinkers may be selected for example frommaleimides, or pyridyldithiols.

The crosslinking may be non-specific by using reactive groups such asaryl azides.

Heterobifunctional crosslinkers are reagents that have different type ofreactive group at either end for example but not limited toamine-to-sulfhydryl or amine-to-carboxyl.

Amine-to-Sulfhydryl crosslinkers may have NHS esters and maleimides ateach end, or NHS esters and pyridyldithiols at each end.

According to some specific embodiments, the heterobifunctionalcrosslinkers may bind the primary NH₂ groups on the BSA surface, and theaptamer via the SH tail.

Examples of heterobifunctional crosslinkers that can bind amine andSulfhydryl groups are selected from but not limited to N-Succinimidyl3-[2-pyridyldithio]-propionate (SPDP),Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), orSuccinimidyl-4-(p-maleimidophenyl) butyrate (SMPB).

Following the first binding agent binding to the matrix, further stepsare preferably performed prior to the target molecule binding anddetection in order to block any reactive group on the matrix surface,with which the chemically reactive groups on biotinylated polymer mayinteract.

In some embodiments, the blocking agent is thereby blocking theremaining free reactive groups on the matrix such as but not limited toprimary NH₂ groups, SH groups and carboxyl groups.

For example, blocking agents such as DMP, Citraconic Anhydride (CA),Sulfo-NHS-Acetate glutaraldehyde, photo-reactive groups,N-acetylcysteine and N,N′-Methanetetraylbis (2-propanamine) (DIPCDI) maybe added. These agents will bind and block for example primary NH₂groups, preferably without affecting the protein charge or hydrophilicand/or hydrophobic state.

In some embodiments, DMP or CA may be used as blocking agents.

According to this embodiment, free DMP having unbound moieties isblocked by the addition of primary NH₂ compounds such as TRIS and Urea.

As can be appreciated, following first binding agent or aptamer bindingto the matrix and the chemical blocking, there are no free unboundreactive groups such as primary NH₂ groups or SH groups on the formedmatrix-first binding agent or matrix-aptamer complex.

Hence upon incubation with the antigen containing sample (as will bedescribed in detail below), the only unbound reactive groups being forexample primary NH₂ groups will be those of the antigen.

Following the formation of the matrix-first binding agent ormatrix-aptamer complex a universal detection probe is added as detailedbelow.

According to some embodiments, the present invention provides methodsfor detecting the presence of a target molecule by using at least oneaptamer.

As detailed above, a single type of aptamer may be used (referred hereinas “single binding agent assay” or “single aptamer assay”), preferablyused in the detection of soluble antigens such as proteins.

In some embodiments, at least two of binding agents (“two binding agentsassay”) may be used in a sequential manner for the detection of targetmolecules. According to some embodiments, the at least two bindingagents, referred to herein as first binding agent and second bindingagent, are of the same class of agents for example being two aptamers(referred to herein as the “dual aptamer assay”). In some furtherembodiments, the first binding agent and the second binding agent are ofa different class of agents.

According to some specific embodiments, the first binding agent is anaptamer and the second binding agent is an antibody. In some furtherspecific embodiments, the first binding agent is an aptamer, antibody orMIP and the second binding agent is an aptamer.

In some embodiments, the first binding agent and the second bindingagent are directed towards the same target molecule or towards differenttarget molecules. In some embodiments, the first binding agent and thesecond binding agent are targeted to different epitopes on the targetmolecule.

In some specific embodiments, two different aptamers are used (“dualaptamer assay” or “two aptamer assay”).

In some specific embodiments, multiple different aptamers are used.

According to some embodiments, the two binding agents assay ispreferably used for the detection of cell associated antigens.

According to these embodiments, the first binding agent may be used toidentify a population of cells, and a second, different binding agentmay be used to detect a specific target antigen, e.g. a surface protein.

In one embodiment, the second binding agent may for example detect asubpopulation of cells, within the cells which are detected by the firstbinding agent.

Non limiting examples for the use of the two binding agents assay may befor example for the detection of cell surface associated cancer markersor detection of viral markers on host cell surface. In a specificembodiment, the two compartment assay may be used in a CMV antigenemiatest for the identification of CMV infection in organ/bone marrowtransplant patients (Raymond U. et al. CMV Antigenemia Following BoneMarrow Transplantation Risk Factors and Outcomes. The 2000 AmericanSociety for Blood and Marrow Transplantation, 17 (2000): 280-288).

In some embodiments, the sample is incubated with the first bindingagent-coated matrix obtained as described above. The incubation enablesthe target molecule to bind to the first binding agent-matrix complex.

According to the “single aptamer assay” embodiment the target moleculeattached to the at least one aptamer bound to the matrix is furtherincubated in the presence of a water soluble polymer which carries achemical reactive group.

According to some other embodiments of the invention, more than oneaptamer may be used in the assay.

According to the “two binding agents assay” the target molecule attachedto the first binding agent bound to the matrix is further incubated inthe presence of a second binding agent bound to a water soluble polymerwhich carries a chemical reactive group.

In the context of the present invention, the term “polymer” denotes anymolecule consisting of a repeating unit capable of being associated witha member of an affinity couple. In the context of the present invention,the chemical reactive group may be for example a succinimidyl Ester (SE)group (for interacting for example with primary NH₂ on the aptamer).

The term “affinity couple” as used herein denotes any two groups havinga high affinity of interaction namely, binding. Non-limiting examplesfor affinity couples are biotin/avidin, antigen/antibody, MolecularImprinted Polymers/target ligand, protein-A/IgG, ligand/receptor, and anucleic acid molecule/complementary sequence.

It is to be understood that the term “a member of an affinity couple” asused herein denotes one member of the affinity couples exemplifiedabove.

The member of an affinity couple may be bound to the polymer via areactive group. Non limiting examples of a reactive group are primaryamines (NH₂), Sulfhydryls (SH), Carboxyls (COOH), Carbonyls (—CHO) orany other reactive group which is capable of binding a member of anaffinity couple and is directed against the chosen target chemical groupon the antigen surface.

According to some embodiments, the second binding agent is incubatedwith streptavidin (SA) thereby a biotinylated second binding agentstreptavidin (b-binding agent-SA) complex is formed for example in amolar ratio of 1:5, optionally followed by the addition of free biotin(molar ratio of 1:5 to SA) to block any free biotin site on the SA. Thesecond binding agent may be different or identical to the first bindingagent.

In this embodiment, the b-binding agent-SA complex is incubated with apolymer associated with a member of an affinity couple and the polymerfurther having a reactive group thereby forming a second bindingagent-polymer complex

In some further embodiments, the second binding agent binds to thepolymer associated with a member of an affinity couple.

The second-binding agent-polymer complex is then added to thematrix-first binding agent-antigen complex.

In some specific embodiments, b-ds-DNA-SE is added to the complex ofSA-second binding agent being an aptamer for example at a molar ratio of2-5:1 (DNA:SA) as schematically shown in FIG. 3. Theaptamers/SA/b-ds-DNA-SE complex may be further purified on an exchangecolumn, e.g. a sephadex G25 column.

In some embodiments, the water soluble polymer comprises CH₂ groups,organic groups or nucleic acids.

In some specific embodiment the member of an affinity couple is biotin(FIG. 2A).

In some other embodiments, the polymer is a DNA. In some specificembodiments, the polymer is a single stranded (ss-DNA) ordouble-stranded DNA (ds-DNA).

In some aspects wherein the polymer is a DNA, it may be obtained fromany source (e.g. human, animal, bacterial, viral, fungal, plant orsynthetic sources). Preferably, a synthetic DNA sequence is used, inorder to avoid similarity and potential cross reactivity to plant,animal, human, bacteria or viral antigens.

The ds-DNA is inserted into a vector, e.g. a plasmid, for example apGEM-T, (Promega USA) according to Manufacturer's instructions and usingmethods well known in the art.

The constructed plasmid may be subsequently subjected to PCR (polymerasechain reaction) by using for example a forward, T7,5′-NH₂ or SH orbiotin labeled primer and a reveres, SP6 primer. The 3′/5′ direction ofthe SP6/T7 primers can be replaced.

The invention is not limited to a particular size of the ds-DNA. Inparticular embodiments, the size of the ds-DNA molecule may be forexample between about 1 kb to about 5 kb, namely the molecule may be ofany size within that range, e.g. about 3 kb.

In the context of the present invention the term “about” is used todenote an approximated range of more or less 10% of the indicated value.

According to some specific embodiments, the synthesis of the ds-DNA ispreformed in the presence of nucleotides associated with a member of anaffinity couple, using a 5′/3′-modified primer, thereby obtaining ads-DNA molecule associated with a member of an affinity couple.

In a specific embodiment, biotinylated dNTP (nucleoside triphosphates)nucleotides may be added during the PCR process, to create biotinylatedds-DNA. In a specific embodiment, by using the ratio forbiotin/non-biotin NTP of 1:5, about 250 biotin groups may be associatedwith a ds-DNA molecule of about 3.5 kb.

The present invention provides methods for an alternative or an addedsignal amplification step. This signal amplification step may be used inboth the “single aptamer assay” and in the “two binding agents assay”.

In some further embodiments, the ds-DNA molecule further comprises atleast one RNA polymerase promoter sequence. For example, the T7 and/orSP6, M13 RNA polymerase promoter sequence.

According to this embodiment, nucleotides associated with a member of anaffinity couple are added to affect DNA or RNA polymerization, therebyobtaining DNA or RNA molecules associated with a member of an affinitycouple.

In some embodiments, the DNA molecule comprises a reactive group at its5′ and/or 3′ termini. In some embodiments, the reactive group issuccinimidyl ester (SE).

According to these embodiments, SE may be subsequently attached to theds-DNA associated SH group by reacting the biotin ds-DNA with a compoundthat can react with the SH group. For example compounds from themaleimids family may be used such as but not limited toSuccinimidyl-4-(p-maleimidophenyl) butyrate (SMPB),Sulfo-Succinimidyl-4-(p-maleimidophenyl) butyrate (s-SMPB),Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC).

The synthesized product obtained thereby is a ds-DNA carrying asuccinimidyl group (biotin-ds-DNA-SE), which may covalently bind toprimary NH₂ groups on the antigen. A schematic representation of themolecule is shown in FIG. 2B.

According to some specific embodiments, the biotin-ds-DNA-SE maydirectly bind the antigen, which is already bound to the aptamer(through specific aptamer-antigen recognition).

According to a specific embodiment, binding of biotin-ds-DNA-SE to theantigen may be for example by covalently binding the antigen's primaryNH₂ group, preferably from Lysine and/or Arginine residues.

As detailed above, it is appreciated, that all other primary NH₂ groupsfrom the proteins coating the matrix have been pre-blocked (e.g. by theaddition of DMP or CA). The present invention encompasses the use ofdetectable signal amplification methods well known in the art.

In some embodiments, the polymer associated with a member of an affinitycouple and signal detection is obtained by adding a complementary memberof said member of an affinity couple associated with a detectablemoiety.

It is to be understood that the term “a complementary member” as usedherein denotes the complementary member within each couples.

Affinity couple as used herein denotes any two groups having a highaffinity of interaction namely, binding. Non-limiting examples foraffinity couple is selected from the group consisting of biotin/avidin,antigen/antibody, Molecular Imprinted Polymers/target ligandprotein-A/IgG, ligand/receptor, and a nucleic acidmolecule/complementary sequence.

The term “detectable moiety” as used herein denotes any compound capableof producing a detectable signal. According to the present invention,the detectable moiety is selected from a chromophore, a fluorophore or aluminancephore.

Specific examples include but are not limited to enzyme-labeledfluorescence (ELF), Hybridization signal amplification method (HSAM),radioactivity-based hybridization assays.

In some embodiments, the member of an affinity couple is biotin and thecomplementary member of said member is any anti-biotin molecule such asfor example avidin or modifications or derivatives thereof (e.g.streptavidin).

In some embodiments, signal amplification may be achieved byenzyme-labeled fluorescence by adding for example avidin or streptavidincovalent/bound to horseradish peroxidase (HRP) enzyme or alkalinephosphatase (AP) enzyme, (HRP/AP=SA), followed by the addition of therelated substrate and monitoring the signal with the related photometeras demonstrated in FIG. 4A. As appreciated, Alkaline Phosphatase (AP) orHorse Radish Peroxidase (HRP) substrate detection may be achieved bychromatic signal, fluorescence signal or luminescence signal, which maybe detected using various spectrophotometers and fluorometers.

Enzyme-Labeled Fluorescence (ELF) (Paragas V B, Zhang Y Z, Haugland R P,Singer V L. The ELF-97 alkaline phosphatase substrate provides a bright,photostable, fluorescent signal amplification method for FISH. J.Histochem. Cytochem. 45 (1997): 345-57) and Hybridization signalamplification method (HSAM) (Kerstens H M, Poddighe P J, Hanselaar A G.A novel in situ hybridization signal amplification method based on thedeposition of biotinylated tyramine. J. Histochem. Cytochem. 43 (1995):347-52), were developed as an alternative, non-radiation hybridizationbased system for the sensitive and rapid identification of DNA and RNAand semi-quantification in histochemical assays such as: in situhybridization techniques and Northern and Southern blot analyses(Kerstens HM, Poddighe P J, Hanselaar AG. A novel in situ hybridizationsignal amplification method based on the deposition of biotinylatedtyramine. J. Histochem. Cytochem. 43 (1995): 347-52; Peter Birner,Barbara Bachtiary, et al. Signal-Amplified Colorimetric In SituHybridization for Assessment of Human Papillomavirus Infection inCervical Lesions. Mod. Pathol. 14 (2000:702-709). The test uses a methodthat creates a chromic, fluorescence or luminescent signal whosebrightness depends on the amount of nucleic acid present. Test resultsare calibrated in numbers of nucleic acid particle equivalents pertested phase. The ELF test is similar in results but not in technique tothe PCR test (Peter Birner, Barbara Bachtiary, et al. Signal-AmplifiedColorimetric In Situ Hybridization for Assessment of HumanPapillomavirus Infection in Cervical Lesions. Mod. Pathol. 14(2001):702-709).

In general, the enzyme catalyzes a reaction of a substrate whichgenerates light signal (detectable in spectrophotometer, fluorometer orluminometer). The amount of light emitted increases with the amount ofthe specific nucleic acid present in the sample (Paragas VB, Zhang YZ,Haugland RP, Singer VL. The ELF-97 alkaline phosphatase substrateprovides a bright, photostable, fluorescent signal amplification methodfor FISH. J. Histochem. Cytochem. 45 (1997): 345-57).

According to some embodiments, the nucleic acid molecule comprises areactive group and further comprises a polymerase promoter sequence.According to these embodiments, the nucleic acid molecule is notassociated with a member of the affinity couple prior to binding to theantigen (in the “single aptamer assay”) or to the second binding agent(in the “two binding agents assay”). According to some embodiments, inthe “single aptamer assay” the nucleic acid molecule comprising areactive group and further comprising a polymerase promoter sequencebinds to the antigen, for example by binding the antigen's primary NH₂groups, preferably from Lysine and/or Arginine residues.

As detailed above, it is appreciated, that all other primary NH₂ groupsfrom the proteins coating the matrix are blocked by the addition of, forexample DMP.

According to other embodiments, in the “two binding agents assay”,nucleic acid molecule comprising a reactive group and further comprisinga polymerase promoter sequence binds to the second binding agent asdetailed above.

In some embodiments, signal amplification may be achieved by convertingthe nucleic acid being ds-DNA into RNA, by using the T7 or SP6 promotersequences located on the nucleic acid and the addition of nucleotidesassociated with a member of an affinity couple in the T7/SP6 RNApolymerase reaction.

In some embodiments, the RNA or DNA molecules associated with a memberof an affinity couple are contacted with a complementary member of saidmember of an affinity couple associated with a detectable moiety.

For example RNA or DNA molecules are anchored to a matrix coated with acomplementary member of said member of an affinity couple.

In some embodiments, the RNA molecule associated with a member of anaffinity couple may then be transferred to a matrix labeled with acomplementary member of said member of an affinity couple for detectionas schematically shown in FIG. 4B.

In some specific embodiments, the DNA comprising a polymerase promotersequence is incubated in the presence of biotinylated NTPs undersuitable conditions to affect DNA or RNA polymerization.

In some embodiments, the formed biotinylated RNA is anchored to anavidin coated tube or beads with simultaneous or subsequent addition ofHRP or AP.

In some specific embodiments, branched DNA assay may be performed.

As appreciated, branched-DNA is essentially a sensitive hydridizationtechnique which involves linear amplification (Edmonds M. Branched RNA.BioEssays 6: 212-21). The Branched-DNA Signal Amplification Assay is analternative hybridization based system for the sensitive and rapiddetection of nucleic acids (Edmonds M. Branched RNA. BioEssays 6:212-21; Chien-Hung chen et al. Quantitative detection of hepatitis Bvirus DNA in human sera by branched-DNA signal amplification. J. ofvirological methods, 53 (1995):131-137). The test uses a method thatcreates fluorescence/luminescent signal whose brightness depends on theamount of nucleic acid present. The Branched-DNA test is similar inresults but not in technique to the PCR test (Tao Chen, et al. Valuationof Quantitative PCR and Branched-Chain DNA Assay for Detection ofHepatitis B Virus DNA in Sera from Hepatocellular Carcinoma and LiverTransplant Patients. J. of Clinical Microbiology, 38 (2000): 1977-1980).

Several different oligonucleotides are used in a Branched-DNA assay. Inthe first stage, the oligonucleotides are used to capture and encore thetarget nucleic acid onto a solid support. Employing DNA hybridizationtechniques DNA branch units are being built, and HRP or AP strep-avidincomplexes are being associated with the “branch” biotinylatedoligonucleotides, thereby creating a dense decoration of the DNA withthe enzyme, allowing high sensitivity of the assay (Edmonds M. BranchedRNA. BioEssays 6: 212-21).

According to these embodiments, a PCR product is generated as describedabove by using a forward thiol labeled primer and a reverse biotinlabeled primer.

The ds-DNA, PCR product, is separated to single strands, for example byincubating the ds-DNA with strepavidin coated magnetic beads and heatingto 95° C. to enable separation of the two DNA strands. The biotinylatedDNA strand is then removed by precipitating the beads.

The thiol labeled ss-DNA strand is then incubated with s-SMPB or s-SMCC,which reacts with SH residual groups on the ss-DNA strand, to form ass-DNA chain attached to a succinimidyl group—ss-DNA-SE, that can bindto a captured antigen primary NH₂ groups (FIG. 5A). For two aptamersassay, the succinimidyl ester group may bind to a complex of SA-aptamersby the binding of the succinimidyl group from the ss-DNA to the primaryNH₂ groups of the SA. This results in formation of a complex ofaptamer-SA-ss-DNA (as schematically shown in FIG. 5B).

It is noted that the ss-DNA is anchored to the beads as it is covalentlybound to the antigen, therefore, the first stage of the DNA branchtechnique is eliminated, i.e. anchoring the ssDNA to the matrix bymultiple primer hybridization and full DNA sequence can be used forbranched associate hybridization.

Next, using LNA (locked nucleic acid), in order to strengthen thehybridization, a Branch unit is constructed. See schematicrepresentation in FIGS. 5C and 5D. According to one aspect of theinvention, the detection methods provide a qualitative or anon-quantitative assessment of the presence of the target antigen.

When employing SA/Aptamers/b-DNA complex in certain embodiments, such asdetecting cell-surface antigens (for example bacterial surfaceantigens), gravitation (e.g. centrifugation) may be used for cellseparation and washing and thereby avoiding the use of an additionalcapture particle bead or surface as described above.

In general, 5′ labeled Biotin, NH₂ and SH primers and Aptamers arecommercially available. Reaction buffers are selected according tomanufacturer's suggestion in each stage. Commercially available PCRcompounds, Magnetic beads, cross linkers, SA, SA=AP/HRP and AP/HRPsubstrate are used according to the manufacturer's instructions withrequired modifications, as detailed. Modified Branch-DNA assay isconstructed according to the selected DNA sequences.

In one specific embodiment, the present invention provides a method forthe detection of a target molecule in a sample comprising:

a. obtaining at least one aptamer capable of binding to said targetmolecule, wherein said at least one aptamer is bound to a matrix;

b. incubating said at least one aptamer which is bound to the matrixwith the sample under conditions allowing the binding of the aptamer tothe target molecule; thereby forming a matrix-aptamer-target moleculecomplex;

c. contacting the matrix-aptamer-target molecule complex formed in step(b) with a biotinilated nucleic acid molecule wherein said biotinilatednucleic acid molecule further comprising SE; thereby forming amatrix-aptamer-target molecule-biotinilated nucleic acid complex; and

d. contacting said complex obtained in step (c) with streptavidinassociated with HRP or AP and a suitable substrate,

wherein the amount of detectable signal produced by the substrate isindicative of the presence of said target molecule in the sample.

In accordance with a further aspect, the present invention provides akit comprising

-   -   (a) at least one aptamer; and    -   (b) a polymer wherein the polymer is associated with a member of        an affinity couple and wherein said polymer is further        associated with a reactive group.

In accordance with some further aspect, the present invention provides akit comprising

-   -   (a) at least one aptamer; and    -   (b) a nucleic acid molecule comprising a reactive group and        further comprising a polymerase promoter sequence.

In some embodiments, the kit is provided herein is for use in a “singleaptamer assay”.

In accordance with yet a further aspect, the present invention providesa kit comprising

-   -   (a) a first binding agent; and    -   (b) a second binding agent—polymer complex, wherein the polymer        is associated with a member of an affinity couple and wherein        said polymer is further associated with a reactive group.    -   In accordance with yet a further aspect, the present invention        provides a kit (a) a first binding agent; and    -   (b) a second binding agent-nucleic acid complex, wherein the        nucleic acid in said complex comprises a reactive group and        further comprises a polymerase promoter sequence.

In some embodiments, the kit provided herein is for use in a “twobinding agents assay”.

According to some embodiments, the polymer is a nucleic acid.

According to some embodiments, the polymer is biotinylated.

According to some embodiments, the active group is a succinimidyl estergroup.

According to some embodiments, the aptamer is bound to a matrix.

According to some embodiments the first binding agent is bound to amatrix.

In some embodiments, each of the first or second binding agentsindependently is selected from the group consisting of an aptamer, anantibody, a receptor ligand or a MIP.

The kits according to the invention may comprise reaction buffers and/orwashing buffers and/or instructions for use.

In some further embodiments, the nucleic acid is not associated with amember of the affinity couple. In some specific embodiments, the nucleicacid is a non biotinylated ds-DNA.

In some embodiments, the polymer is associated with a member of theaffinity couple. In some specific embodiments, the polymer is abiotinylated ds-DNA.

In some further embodiments, the ds-DNA comprises RNA or DNA polymeraseenzyme.

The kits according to the invention may further comprise RNA or DNApolymerase enzyme, NTPs, and NTPs associated with a member of anaffinity couple. The member of an affinity couple is for example biotin.

The kits may also comprise at least one detection enzyme and optionallya substrate for said detection enzyme, which is HRP or AP.

FIG. 6 shows a schematic illustration of the “single aptamer assay”.

In accordance with one example of the single aptamer assay, the aptameris covalently bound to the matrix, for example to magnetic beads, and isfurther used to detect target antigen.

In a non limiting example of the present invention, the assay comprisesthe following steps:

In the first step, a complex comprising a matrix (which may bepre-coated as described above), for example magnetic beads, is bound toan aptamer, possibly a biotinylated aptamer and is incubated with a testsample allowing the aptamers to bind to the tested antigen. The matrix,now containing the bound antigen, is obtained from the sample.

In the second step for example a biotinylated ds-DNA-SE is added andcovalently binds the antigen.

In some specific embodiments, if the ds-DNA-SE is not biotinylated, aT7/SP6 RNA polymerase amplification reaction is performed at this step,preferably in the presence of biotinylated dNTPs resulting in obtaininga biotinylated RNA molecule, as described above, followed by binding thebiotinylated RNA molecule onto a SA matrix. Alternatively, ss-DNAcomplexes arebound to the antigen.

In the third step, a modified Enzyme-Labeled Fluorescence SignalAmplification step is performed, by adding SA=HRP/AP reagent, followedby the addition of the related substrate.

In one embodiment wherein ss-DNA is used, a Branched DNA complex isformed before adding the SA=HRP/AP reagent.

In some specific embodiments the first two steps or at least the secondstep may be skipped. In such embodiments, the aptamer-SA-bDNA complex isused.

In some embodiments, all the steps are performed in a single test tube.

In some other embodiments, the assay is performed in several test tubes.For example, steps 1 and 2 including the RNA amplification are performedin one Tube and step 3 is performed in another tube.

According to some embodiments of the invention, step 2 of the assay canbe performed in two main formats: using ds-DNA or ss-DNA. In embodimentswherein ss-DNA is used, the RNA polymerase step is eliminated, and astep of “branched” DNA formation may be added. It is noted that in thecase of RNA amplification, The RNA can be bound to SA-magnetic beads (orother matrix), and a branched-RNA assay can be performed.

In accordance with the “single aptamer assay” embodiment, the presentinvention provides means to detect and quantitate (or semi-quantitate) asoluble antigen such as a protein or any other antigen which carries areactive group, e.g. free primary NH₂ groups. Similarly any otherchemical group can be used in the context of the present invention,adapting accordingly the reactive group on the polymer and thechemically blocking reagent for the matrix.

In addition, the present invention provides a tool to detect thepresence of a virus or bacteria, in a sample.

According to the present invention at least one aptamer is used in thedetection assay.

In a specific example for the “single aptamer assay”, the kit comprises:

(a) an aptamer bound to a coated matrix and (b) a biotinylated or nonbiotinylated ds-DNA associated with a succinimidyl group. The kitoptionally further comprises related reaction and washing buffers.

The kit may comprise also a positive control antigen.

In addition, the kit may comprise a T7 RNA polymerase enzyme, NTPs,biotinylated UTP and related buffers.

The kit may further comprise biotinylated RNA bound to a capture matrix,such as beads.

The kit may further comprise SA=HRP/AP solution.

The kit may further comprise HRP/Alkaline Phosphates substrate buffersand stoppers.

In accordance with the “single aptamer assay” embodiment of theinvention, the following assay is an example for the assay which may beperformed using the kit of the invention:

-   -   1. A test sample is added to a pre-coated matrix-aptamer        complex. The sample is incubated with the pre-coated        matrix-aptamer complex for about 30-60 min at room temperature        (RT) followed by magnetic separation and washing (×5 with        PBS/EDTA 1 mM/Tween-20 0.01%).    -   2. biotin-ds-DNA associated with a Succinimidyl group is added        and incubated for about 30 min at RT followed by magnetic        separation and washing (×5 with PBS/BSA 2%/Tween-20 0.01%).    -   3. Optionally, a T7 RNA polymerase reaction is performed by        adding the components required for the reaction such as: T7 RNA        polymerase enzyme, NTPs, biotinylated UTP and related buffers        for 60 min at 37° C. followed by magnetic separation and        transfer of the RNA to the capture tube.    -   4. SA=HRP/AP solution is added for an incubation of 30 min at RT        followed by washing (×5 with PBS/BSA 2%/Tween-20 0.01%).    -   5. The related substrate solution is added for 5-10 min at RT        followed by measurement of absorbance or fluorescence (such as        TMB as chromophore for HRP, pNPP as chromophore for AP, Attopose        as fluorophore for AP).

Two additional tests may be performed in parallel, the first assay is anegative control assay including only the buffers and washing solutionsand the second assay is a positive control which contains apre-determined known amount of antigen in the sample.

If the target antigen (Ag) is present in the tested sample, it willinduce Succinimidyl-dsDNA complex binding to the magnetic beads via theAptamers Ag complex. This will allow the binding of the SA=Enzyme to thebiotinylated DNA and eventually induce a measurable signal (with andwithout additional RNA transcription).

If the antigen is not present in the sample, then there will be nosignal detected in both negative control and sample tubes.

This experiment may provide the following:

-   1. A qualitative (yes or no) test—by comparison with a negative    control tube.-   2. Semi-quantitative test—by adding control samples that contain low    and high antigen concentration.-   3. Quantitative test—by adding control wells that contain full Ag    concentration standards.

According to some aspects of the invention, it is appreciated that whenthe target is a cell surface antigen, the first binding agent (beingbound to the matrix) is used for the identification of the target cells,and a second binding agent is used for identification of a subpopulationcomprising for example a surface antigen. FIG. 7 shows a schematicillustration of the “two binding agents assay”. For example, twoaptamers may be used in the detection assay. In such case each of theaptamers may be directed to a different target protein or to a differentepitope on the same protein. The dual aptamer detection assay may besuitable for example for the detection of a cell surface molecule, orfor the identification of specific cell populations or pathogens.

According to some embodiments, the present invention provides a kit fordetection of an antigen using a “two binding agents assay”. This assaymay be used to identify an antigen which is associated with otherantigens, in the biological sample, such as cell surface antigens andcomplexes.

Thus, for example in the “two binding agents assay” embodiment, theds-DNA may be bound to a streptavidin associated aptamer complex,directly to an aptamer. In some specific embodiments, the ds-DNA may bebound to a streptavidin associated aptamer complex to a secondaryrelated antibody

For example in the “two binding agents assay”, the kit may comprise:

-   -   a. an aptamer bound to a coated matrix; and    -   b. a second Aptamer or Aptamer/SA associated with a biotinylated        ss-DNA or a ds-DNA chain in solution. The it optionally further        comprises related reaction and washing buffers.

The kit may further comprise a positive control antigen.

The kit may further comprise a T7, SP6, or M13 RNA polymerase enzyme,NTPs, and optionally, related buffers.

The kit may further comprise Biotinylated NTPs.

The kit may further comprise biotinylated RNA bound to a capture matrixsuch as for example beads.

The kit may further comprise SA=HRP/AP in solution.

The kit may further comprise HRP/Alkaline Phosphates substrate buffersand stoppers.

It is appreciated that according to the invention, any fluorometricsubstrate may be used in accordance with the invention, for example, thecolor based Alkaline Phosphate substrate pNPP, a fluorophor substratesuch as AttoPhos®, AP Fluorescent Substrate, Adamantyl-1,2-dioxetanephosphates or Lumi-Phos 530. The use of such fluorescence substrateswill allow increasing the assay sensitivity, for the detection of as lowas 10³ Ag copies in the assay tube.

The measurement of the fluorescence in accordance with the invention maybe performed with any fluorescence measurement device including Handheld, portable fluorometer devices such Invitrogene's—“Qubit®Fluorimeter”, Topac Inc.'s—“picofluor” and Promega's—“TBS-380 MiniFluorometer”.

These devices are highly sensitive and can detect fluorescence andillumination at vireos excitation and emission wave lights. The use ofsuch devices will allow performing the assay at point of care (POC).

EXAMPLES Example 1 Preparation of Aptamers Associated with a Matrix andHaving Blocked NH₂ Groups A) Use of Streptavidin (SA) DynaBeads (DB) andBiotinylated-Aptamers

Streptavidin DynaBeads M-280 (Invitrogene) were washed with Tris/EDTABuffer (TE) and incubated with commercial C₁₂-biotinylated aptamers,according to manufacturer's instructions.

At the end of the incubation period, biotin, at double molar ratio tothe beads capacity was added, to block any free SA sites. Followingwashings with TEN (TE with 1M NaCl) and phosphate buffered saline (PBS)buffers, the DB-SA-aptamer complexes were kept at 4° C. in PBS.

For the blocking of all primary NH₂ groups, on the SA surface, theDB-SA-aptamer complexes were incubated with 5 mg/ml dimethylpimelimidate (DMP) cross linker (Pierce) in Borate buffer, as directedby the manufacturer, for overnight at RT. It is noted that citraconicanhydride (CA) can also be used for the same purpose—see below).

To block unbound DMP reactive sites, the beads were washed (Boratebuffer), and incubated in borate buffer which contains Tris (100 mM) for6 h at RT. Following washings with PBS buffers, the preparedDMP/DB-SA/Aptamers were kept at 4° C. in PBS/EDTA.

B) Use of BSA Coated DynaBeads and SH-Aptamers—Association ViaCross-Linking

DynaBeads M280 Tosylactivated (10 mg) were coated with BSA according tomanufacturer's instructions, using excess BSA. Blocking of theTosylactivated residues on the beads is done by using Tris.

DB-BSA were incubated for 1 h at RT withSulfo-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(s-SMCC) or Sulfo-Succinimidyl-4-(p-maleimidophenyl) butyrate (s-SMPB)cross linkers (50 μl of 20 mM in DMSO into 500 μl of 1 mg beads inPBS/EDTA), followed by 5 washes in PBS/EDTA.

In parallel, SH-aptamers were obtained by either treating thetiol-Aptameres with DTT (10 mM, 30 min at RT), or by creating a SH-Groupon the NH₂-aptamers as follows: 50 μl of the aptamer-NH₂ (200 mM) wereincubated at RT for 45 min with 5 μl of N-Succinimidyl3-[2-pyridyldithio]-propionate (SPDP) cross linkers (20 mM) in a totalvolume of 50 μl in PBS/EDTA. 5 μl of DTT 1M was added and samples wereincubated for 30 min at RT. Excess SPDP and DTT were removed by using 1ml Sephadex G-25 dray column.

Immediately 50 μl of the aptamer-SH were added to the activated DB-BSA(5 mg beads in 200 ul of PBS/EDTA) and incubated for 2 h at RT.

Acetyl-Cys (100 μl, 50 mg/ml in PBS-EDTA1 mM buffer) was added andincubated for 6 h at RT (for blocking any remaining N-maleimidomethylreactive groups, of the cross-linker).

Alternatively, the BSA coated beads were incubated with the Aptamer-NH₂as describe above, in the presence of 25 μl of DMP cross-linker (5mg/ml) over night at RT, followed by at least 6 h incubating with 0.2MTris (pH-7.4).

After three washings with PBS/EDTA and additional two washings with 150mM phosphate buffer pH=9.0, 250 μl of citraconic anhydride 20 mM in 150mM phosphate buffer ph=9.0 were added and samples were incubatedovernight at RT. Samples were washed five times with PBS/EDTA and keptat 4° C. in PBS/EDTA.

Alternatively, after three washings with PBS/EDTA and additional twowashings with Borate buffer, the aptamer associated beads were incubatedwith 50 μl of 5 mg/ml DMP over night at RT in a total volume of 300 μlin Borat buffer. Samples were washed five times with PBS/EDTA and keptat 4° C. in PBS/EDTA

C) Preparation of Biotinylated ds-DNA Attached to Succinimidyl Ester(‘b-ds-DNA-SE”)

A plasmid carrying the Hepatitis B scrambled sequence was prepared byfishing out the DNA from HBV plasmid PadwHTD (Giladi H, Ketzinel-GiladM, Rivkin L, Felig Y, Nussbaum O, Galun E. Small Interfering RNAInhibits HBV Replication in Mice. Molecular Therapy, 8 (2003):769-76)and inserting it into pGMT-eazy, to receive Plasmid pGM-HBD (FIG. 8).

Associated forward primers: (SEQ ID NO. 1)T7 - 5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO. 2)B5 - 5′-CCTCTGCCGATCCATACT GCGGAAC-3′ (SEQ ID NO. 3)B1 - 5′-GGAGGCTGTAGGCATAAATTGGTCTGCGC-3′ Associated reverse primers:(SEQ ID NO. 4) SP6 - 5′-ATTTAGGTGACACTATAGAA-3′ (SEQ ID NO. 5)B10 - 5′-ATGCGCTGATGGCCTATG G-3′ (SEQ ID NO. 6)B2 - 5′-CCCGAGATTGAGATCTTCTGCGACGCGGCGATTGAGACC-3

pGM-HBD was submitted to PCR as follows: 1× Pentium-Taq Buffer(Invitrogen), 1× Pentium-Taq MgCl solution, 200 μM each dNTP, 50 μMbiotinylated-dCTP, 50 pm of each primer (SP6/T7), 5 ng Template and 1 μlof Pentium-Taq, in 100 μl H2O.

If not state otherwise, the forward primer used was Tiol-C₁₂-T7 primer(Amine-C₁₂-T7 or C₆ primers also be used).

PCR instrument program was designed according to the Pentium-Taq manualsfor a PCR product of 3.5 Kb.

The Tiol labeled biotinylated PCR products were pooled, treated with 50mM DTT for 30 min at RT and DNA-SH was cleaned on MEGAquick-spinE-PCRextraction column or by employing 1 ml Sephadex G-25 dray column (orequivalent). To add a succinimidyl ester group, the biotinylatedds-DNA-SH was incubated with sSMPB/sSMCC cross linker (50 mg/ml, Pierce)for 30 min at RT. biotinylated ds-DNA-succinimidyl ester reagent(b-DNA-SE) was purified by Et-OH precipitation or by employing 1 mlSephadex G-25 dray column. DNA-SE was re-suspended in PBA/EDTA containsPVP20 mg/ml.

It is noted that any other cross linkers that will create an Aminereactive group at the DNA 5′, such as Maleic Anhydride can be used.

D) Preparation of Streptavidin-b-Aptamer Attached to ds-DNA-SE

Streptavidin (SA) was incubated with biotinylated aptamers at a molarratio of 1:4 (1 nm: 4 nm) in a total volume of 80 μl TEN buffer (TE with1M NaCl), for 30 min at RT. Biotin, 2 nm in 20 μl TEN, were added andincubated for 30 min at RT (To block free SA sites).

SA-aptamer complex was cleaned on a sephadex G-25 super fine 5 mlcolumn. Each 25 μl of the above (about 250 pm SA) were incubated withabout 20 μg of b-DNA-SE in PBS/EDTA (1 mM), in a total volume of 100 μl,for 1 h at RT. 10 μl 1M Tris was added (To block the un-bound SE groups)ad incubated for 1 h at RT.

Aptamer-SA-bDNA complexes were diluted 1:10 in PBS 0.01% tween 20.

100 μl reagent was used per assay sample.

E) Preparation of Aptamer Directly Attached to ds-DNA-SE

ds-DNA-SE was incubated with NH₂-aptamers at a molar ratio of 2:1 in atotal volume of 50 μl PBS/EDRA 1 mM for 120 min at RT. Un bound reactiveSE groups were blocked with Tris (0.2M) for 3 h at RT in total volume of100 ul.

Aptamer-bDNA complexes were diluted 1:10 in PBS 0.01% tween 20.100 ul reagent was used per assay sample.

F) Preparation of IgG/b-DNA Complex

IgG, 50 μl of 2 μg/ml, were incubated with 25 μg DNA-SE for 1 h at RT ina total volume of 100 μl PBS/EDTA 1 mM. Tris 1M was added (10 μl) (toblock un-bound SE) and incubated for 1 h at RT.

IgG-bDNA was diluted 1:10 in PBS/tween 20 (0.01%) and 100 ul were usedfor each assay sample.

Example 2 Validation of the Enhancement Capability of b-DNA-SE

The Legend max human TNF ELISA kit (BioLegend) was used as a commercialELISA kit.

1 ml of anti-TNFα antibodies solution (Legend max human TNF ELISAkit—BioLegend) was reacted with b-DNA-SE as described above, therebyobtaining modified anti TNFα antibodies. Per each assay, 110 μl ofIgG-bDNA-SE were used.

The assay was performed in 3 different versions [A, B and C] to allowcomparison:

A) According to Manufacturer's instructions.

B) According to Manufacturer's instructions except that instead of thesecondary anti-TNFα Ab solution provided in the kit, the modifiedIgG-bDNA was added.

C) As in (B) except that after the addition of the modified IgG-bDNA astep of T7 RNA polymerase reaction was preformed, with the addition ofBiotin-UTP. The biotinylated RNA was loaded on Avidin-magnetig beads(DB-M280-SA), followed by washing with the washing solution and theaddition of SA-HRP. Beads were washed with the Kit's washing solutionand the kit's substrate was added.

Results:

FIG. 9 clearly shows that the modified Abs (B) gave a result which isabout one and a half logs more sensitive than the unmodified antibodies(A). T7 RNA amplification (C) enhanced the signal, increasing thesensitivity by at least about one more log. In all 3 assays thebackground level was very low if any.

These experiments demonstrate the ability of the multi biotinylated DNAto highly increase ELISA sensitivity, with the possibility of furtherincreasing sensitivity by employing the RNA amplification techniques.This assay is reaching a sensitivity of below 5×10^(e5) TNF moleculesper assay, using a spectrophotometer.

Example 3 Detection of PDGF-BB Using Aptamers

The following Aptamer-based assay was developed for the monitoring ofplatelet-derived growth factor B-chain homodimer antigen (PDGF-BB) inPBS, FCS, human serum and Plasma.

Methods: Reagents:

RayBio Human PDGF-BB ELISA Kit [Cat#: ELH-PDGFBB-001] and its reagentswere used as a reference for the assay.

The aptamers for platelet-derived growth factor B-chain homodimerantigen PDGF-BB (Sigma) were designed as follows as described (Yuan Li,et al. Ultrasensitive Densitometry Detection of Cytokines withNanoparticle-Modified Aptamers. Clinical Chemistry 53 (2007):6; YongHuang, et al. Electrochemical immunosensor of platelet-derived growthfactor with aptamer primed polymerase amplification. AnalyticalBiochemistry 382 (2008): 22).

PDGF-BT-1: (SEQ ID NO. 7)biotin-5′-GCGATACTCCACAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3′ SCREM-BT-1:(SEQ ID NO. 8)biotin-5′-GCGATACTCCACAGCTGACGGCACGGTAAGCATCACCATGATGTCC-3′ PDGF-BT-2:(SEQ ID NO. 9)biotin-5′-GCAGTTACTCAGGGCACTTGCAAGCAATTGTGGTCCCAATGGGC TGAGTAT-3′

DB-SA/Aptamer was prepared as described above, employing the aptamerPDGF-BT-2 and its scrambled form SCREM-BT-1.

Aptamer-SA-bDNA was prepared as described above, employing the aptamerPDGF-BT-1 or SCREM-BT-1.

Anti-PDGF-BB-bDNA-SE complex was prepared as described above.

Assay Construction:

DB-SA carrying the PDGF-BT-2 or SCREM-BT-1, were washed three times andresuspended in PBS/Tween-20 (0.01%).

Recombinant PDGF-BB was added (1000 of 1 pg/ml, 100 fg) in the samebuffer or in fetal calf serum (FCS) 0.01% tween-20, and incubated for 1h at RT while rotating. In a parallel assay, a sample of human-serumcontaining 0.01% tween 20 was added and incubated as above.

Antigen associated DB-SA with the attached aptamers were incubated witheither PDGF-BT-1 or SCREM-BT-1/SA-bDNA-SE or anti-PDGF-BB-bDNA. Afterabout 1 h incubation at RT, the complexes were washed ×3 withPBS/BSA/Tween and then SA-HRP (1:5000 in PBS/BSA/Tween buffer) was addedand incubated for 30 min at RT.

The HRP labeled beads were washed ×5 with PBS/BSA/Tween, and substrate(100 μl) was added to the pellets. The reaction stopper solution wasadded within 2-5 min, beads were removed and supernatants were evaluatedat 450 nm, employing Tecan fluorometer/spectrophotometer.

Results:

The ability of the PDGF2 aptamer to recognize and bind recombinant andnative PDGF-BB to the beads was studied by employing a rabbit antiPDGF-BB antibody which carries a bDNA (FIG. 10A).

It was found that a relatively high signal was detected when a PDGF-BBspecific aptamer (PDGF2) was associated with the beads. Low signal, ifany, (under 0.150D) was detected when the scrambled aptamer wasassociated with the beads.

These results demonstrate the specific binding of the antigen, namelyPDGF-BB, to the beads-aptamer complex.

The results in FIG. 10A also show the ability of the aptamer to bind therecombinant FDGF in serum environment (FCS), as well as to bind nativeFDGF-BB from human sera.

The RayBio Human PDGF-BB ELISA Kit has a sensitivity of 50 fg/assay,showing an average of 0.3 OD for 100 fg antigen, the amount of Ag usedin the experiment. We have demonstrated that by associating to theantibody a large amount of Biotin, and therefore a large amount of HRP,the signal is strengthened thereby enhancing the assay sensitivity.

In addition, a second aptamer was used in order to introduce and bindSA-HRP-bDNA to the Ag/Beads complex.

The results which are represented in FIG. 10B demonstrate thespecificity of the second aptamer PDGF1 to the Ag, i.e. PDGF-BB (Leftside), showing only low background, using the scrambled Aptamer screm1as a second aptamer (right panel)

A relatively high signal, which implies a higher sensitivity, wasobserved when a large number of HRP molecules (about 250) wereassociated with the Aptamer/SA (or IgG) complex, via the attachedbiotinylated ds-DNA. As shown above, the scrambled aptamer failed tobind PDGF, either recombinant or native, to the beads.

Example 4 The Use of Aptamers and b-DNA-SE as a Tool for AntigenDetection and Quantitation—Comparison of Different Amplification Methods

An assay, based on DB-Streptavidin aptamers and biotinylated-DNA-SE, wasdeveloped for the monitoring of PDGF-BB in PBS, FCS and human serum andPlasma.

Materials:

Using the same three aptamers for PDGF-BB as described above,DB-SA/aptamer complexes and b-DNA-SE was prepared as described above.

The DB-SA/aptamers complex was treated with DMP in order to block allprimary NH₂ groups on the SA surface, as described above.

The experiments were performed using 1 pg of PDGF in b-DNA-SE assay and50 fg in T7 RNA polymerase amplification assay.

Results:

The Succinimidyl Ester from the ds-DNA complex is capable of attackingand covalently binding primary NH₂ groups

Since the NH₂ groups on the Streptavidin (SA) associated with the beadsare blocked by the addition of DMP, the only source for primary NH₂groups in the complex is from the PDGF-BB antigen. Thereby ensuring aspecific binding of the ds-DNA-SE only to the antigen, namely PDGF andeliminating any unspecific binding of the ds-DNA-SE to the beads.

Indeed, the results in FIG. 11 suggest that all the SA molecules on thebeads carrying primary NH₂ groups are blocked, and that under the aboveconditions, there is no unspecific bindings of Ag to the beads/aptamerscomplex.

This was also evident when BSA was used as antigen instead of thePDGF-BB antigen.

In addition, as shown in FIG. 11A, the DNA-SE binds only to beadscarrying the PDGF2 aptamers, and thus carrying the PDGF-BB and not tobeads carrying the scrambled aptamer (scrm1).

Moreover, no background was detected neither when PDGF-BB was present inFCS nor in its native environment i.e. in human serum. These resultsindicate the specificity of the above described aptamer assay.

The assay described above also shows high sensitivity, in comparisonwith the RayBio Human PDGF-BB ELISA Kit.

The assay sensitivity can be increased, by performing the T7 RNAamplification step, as shown in FIG. 11B.

As shown in FIG. 11B, the T7 RNA amplification step increased thesensitivity compared to the b-DNA-SE assay by about 1.5 logs.

It is noted that while the RayBio Human PDGF-BB ELISA Kit has asensitivity of 50 fm Ag (about 0.3 OD), the T7 RNA amplification stepmeasured a signal of over 2.0 OD at the same Ag amount in the assay,showing a potential to about 2-3 logs less Ag than with the kit.

Example 5 The Use of ss-DNA-SE and Branched-DNA Assay

In this example, an assay, based on DB associated aptamers and ss-DNA-SEand Branch-DNA assay, were developed for the monitoring of PDGF-BB inPBS.

Materials:

Using the same three aptamers for PDGF-BB as described above,DB-SA/aptamer complexes and b-DNA-SE were prepared as described above.

The DB-SA aptamers complexes were treated with DMP in order to block allprimary NH₂ groups on the SA surface, as described above.

ss-DNA-SE was bound to anti-PDGF-BB as described in experiment 1.

Branch-DNA assay kit for HBV V-3.0 (VERSANT) was used according to themanufacturer's instructions, albeit, eliminating the “DNA capture” step.

After establishing the AP/DNA complex, PNPP substrate was added.

The experiment was performed using the indicated amounts of Ag in assay,as described in example 4.

Assays Samples and Results:

The Branched-DNA assay (which is originally used in the prior art tomonitor and quantitate DNA and RNA) was adapted and found to beeffective and sensitive for the determination and quantitation of aprotein Antigen in the constructed assay, e.g. the detection of PDGF.

As described above, the presented results also show that the ssDNA-SEand the ssDNA-IgG complexes bind only to beads associated with the PDGF2aptamer, and therefore to PDGF, and not to beads carrying the scrambledaptamer (scrm1), indicating the specificity of the assay (FIGS. 12A and12B).

Example 6 The Use of Fluorimetric Base Dye and the Qubit® Fluorometerfor Measurements of BD-Aptamers and b-DNA-SE Based Ag Detection andQuantitation Assay

AttoPhos Substrate is an Alkaline Phosphate fluorimetric substrate (ex.−430 nm/em. −560 nm). In this example the sensitivity of the constructedassay was studied, employing color base Vs flourimetric substrates(PNPP/AttoPhos).

The tested assay was based on DB-SA-aptamers and biotin-ds-DNA-SE, fordetecting PDGF-BB, and using commercially available SA=AP (alkalinephosphates) complex.

Reagents and Experimental Methods:

PDGF-BB antigen, DB-SA/PDGF2 aptamer, and b-DNA-SE were prepared asdescribed in the example above. The DB-SA/Aptamers complex was treatedwith DMP in order to block all primary NH₂ groups on the SA surface, asdescribed above. All other experimental procedures were performed asdescribed above.

The assay was performed as described above employing SA=AP, followed bythe related substrate addition and signal readings.

For Qubit® Fluorometer readings, the samples were first read by Tecanfluorimeter and then transferred to 0.5 ml tube for Qubit® Fluorometerreadings. Qubit® Fluorometer calibration (employing the Quant-iT™ DNAAssay setting), was performed using the “no Ag” sample as “standard #1,and the 100 fg Ag as standard #2. Results are expressed at “related DNAvalues” in ng/ml.

Results:

The results in FIGS. 13A and 13C clearly demonstrate that using theAtto-Phose substrate higher sensitivity of the assay is achieved, about2 logs more sensitive than with the PPNP substrate (10 ag Ag Vs 1 fg Ag,correlated to 4×10² Vs 4×10⁴ Ag molecules in the assay).

The results also demonstrate that the Qubit® Fluorometer can be use as ahand size fluorometer in the assay of the invention (FIG. 13B). Theresults demonstrate that the Qubit instrument is suitable for Atto-Phosefluorescence readings, with sensitivity similar to the Tecanfluorometer.

Example 7 The Use of DB-BSA-Aptamers as a Tool for Antigen Capture andb-DNA-SE for Ag Detection and Quantitation

Cytomegalovirus (CMV) disease is a major life-threatening complicationin recipients of blood and bone marrow transplants (BMTs). CMV may causedeadly interstitial pneumonitis, esophagitis, gastritis, colitis,hepatitis, fever, leukopenia, and a severe wasting syndrome. Identifyingthe CMV infection, combined with ganciclovir/immunoglobulin (IVIg)therapy, is the most effective preventive therapy in BMT patients.Therefore, patients are undergoing surveillance tests (cultures,serology, and antigen detection) of blood and other body fluids todetect evidence of CMV activation.

An assay, based on DB-BSA-Aptamers and biotinylated-DNA-SE, wasdeveloped for the monitoring of the Cytomegalovirus gB surfaceglycoprotein (CMV-gB) in PBS, and human serum and Plasma.

Materials:

Aptamers for CMV-gB were designed as follows as described (Wang, J. etal In vitro selection of novel RNA and DNA ligands that bind humancytomegalovirus and block viral infection, RNA, 6, (2000):571-583,2000).

gB1 (SEQ ID NO. 10)Tiol-C12-5′-TTACGGTCACCTTACCCCTGGGTGTGCTCT TC CCGGTGGG-3′ SCREM-1:(SEQ ID NO. 11)Tiol-C12-5′-GCGATACTCCACAGCTGACGGCACGGTAAGCATCACCATGATGTCC-3′

DB-BSA/Aptamer was prepared as described above, employing CA for thematrix primary NH₂ blocking.

DNA-SE was constructed as describe above.

Assay Construction I:

DB-BSA carrying the different aptamers were washed, re-suspend inPBS/Tween-20 (0.01%) and then reformed into a pellet. To a pellet of 0.5mg beads, recombinant CMV-gB or PDGF-BB were added (100 μl of 10 pg/ml)in PBS/Tween-20 (0.01%) and 1% BSA and incubated for 1 h at RT withrotating.

Samples were washed five times with PBS-EDTA-Tween and 100 μl b-DNA-SEwas added and the samples were incubated for 60 min at RT.

Following three washings with PBS-Tween and additional two washings withPBS-BSA-Tween, 100 μl of SA-HRP (1:5000) were added and incubated for 30min at RT.

Samples were washed five times with PBS-BSA-Tween and then 100 μl TMB(TMB One-Step Substrate Reagent, Thermo Scientific) were added andincubated for 10 min at RT.

Reaction was stopped with 100 μl Stop solution and the samples were readat 450 nm within 30 min.

Results:

As shown in FIG. 14, the blocking of the NH₂ groups of the BSAassociated beads with CA was effective, therefore the only source forprimary NH₂ groups in the system is the bound Ag.

As shown in FIG. 14, the b-DNA-SE binds only to beads carrying the gB1aptamer, in the presence of the CMV-gB antigen (14A) and not bind tobeads carrying the scrambled aptamer (scrmb1, 14B).

In addition, signal is obtained only with the CMV-gB antigen, while onlybackground signal is obtained with the PDGF-BB Antigen. These resultsindicate that the gB1 aptamer is specific to the CMV-gB antigen and doescapture the PDGF-BB Antigen, or the buffer associated BSA.

Assay Construction-II:

DB-BSA/Aptamer (pellet of 0.5 mg beads) prepared as described above werere-suspended in cultured CMV (1.4×10e4 PFU) spiked into either PBS/BSAbuffer or into human serum (100 μl). Following 60 min incubation at RTwith rotation, samples were washed five times with PBS-EDTA-Tween and100 μl b-DNA-SE was added. The samples were incubated for 60 min at RT,and then washed three times with PBS-Tween and twice with PBS-BSA-Tween.

100 μl of SA-HRP (1:5000) were added and the samples were incubated for30 min at RT. Samples were washed five times with PBS-BSA-Tween. Then,100 μl TMB were added and the samples were incubated for 10 min at RT.Reaction was stopped with 100 μl Stop solution and the samples were readat 450 nm within 30 min.

As shown in FIG. 15, the b-DNA-SE reagent binds only to beads associatedwith the gB1 aptamer in the presence of the cultured CMV, and not tobeads associated with the scrambled aptamer.

The gB1 aptamer did not bind any protein component in the PBS/BSA bufferor in the tested human serum.

It is noted that another CMV antigen suitable for detection inaccordance with the present invention is the early viral matrixstructural protein pp65 in a subject's leukocytes.

The detection assay of the invention is suitable to determine fewleukocytes carrying the pp65 on their surface, in patient's bloodsample, without the need for using fluorescence microscope or FACStechniques. Using anti leukocytes aptamers bound to magnetic beads,leukocytes are separated and concentrated from whole blood sample.Adding a second biotinylated aptamer, which is directed against thepp65, allows the binding of the streptavidin ds-DNA. First amplificationoccurs as there are several hundred to several thousand copies of pp65per cell. Second amplification occurs during the T7 RNA polymerasereaction (about 2 logs of bound ds-DNA). Without wishing to be bound bytheory, one infected cell (in 0.1-1.0 ml of whole blood sample) mayinduce about 10⁵ RNA copies for bRNA reaction, which is sufficient fordetection.

Example 8 Capture and Quantitation of Listeria Bacteria by Two Aptamers,Employing DB-BSA-Aptamers and b-DNA-SE

An assay, based on DB-BSA-aptamers and b-DNA-SE, was developed for themonitoring and quantitation of the Listeria bacteria in PBS and inswabs.

Reagents:

Aptamers for Listeria were designed as follows as described in patentapplication US 20090203028.

Ap03: (SEQ ID NO. 12) NH₂—C₁₂-5′-ATCGATGATCTGGTCGCCGTAACACTACCCACATATACGACCAGG-3′ Ap08: (SEQ ID NO. 13)NH₂—C₁₂-5′-ATCCATGGGGCGGAGATGAGGGGGAGGAGGGCGGGTAC CCGGTTGAT-3′

Aptamer NH₂—C₁₂-SCREMB-1 was prepared as described above.

The b-ds-DNA-SE used in this Example was constructed as describe above.

DB-BSA/Aptamer preparation—Use of BSA coated DynaBeads andNH₂-C12-5′-Aptamers.

Dyna-Beads M280 Tosylactivated (10 mg) were coated with BSA according toManufacturer's instructions, using excess BSA (“DB-BSA”). Blocking ofexcess BSA was done by adding Tris.

DB-BSA were incubated over night at RT with 50 μl of one or more of thethree aptamer-NH₂ described above (200 mM), and 75 μl of DMPcross-linker (1 mg/ml), in a total volume of 300 μl in Borate Buffer.

Free primary HN₂-groups on the BSA were then blocked by the addition of100 μl of DMP cross-linker (5 mg/ml in Borate Buffer) and let over nightat RT

Free active ends of the DMP were blocked with Tris as described above.Samples were washed five times with PBS/EDTA and kept at 4° C. inPBS/EDTA.

Assay Construction:

DB-BSA-aptamer preparations were washed, re-suspend in PBS/Tween-20(0.01%) and aliquoted as needed. To a pellet of 0.5 mg DB-BSA-aptamercomplexes, varying amounts of several Listeria strains were added (in100 μl of PBS/Tween/BSA). The samples were incubated for 1 h at RT byapplying constant rotation. Samples were then washed five times withPBS-EDTA-Tween and 100 μl of b-DNA-SE preparation were added andincubated for 60 min at RT.

Following three washings with PBS-Tween additional two washings withPBS-BSA-Tween, 100 μl of SA-HRP (1:5000) were added and incubated for 30min at RT. Samples were washed five times with PBS-BSA-Tween and 100 μlTMB were added and incubated for 10 min at RT. Reaction was stopped with100 μl Stop solution and samples were read at 450 nm within 30 min.

Results:

As shown in FIG. 16A, the aptamer Ap03 recognized all 4 Listeria strainswhile the aptamer Ap08 did not recognize the LS3 strain. Both aptamersdo not recognize the E. Coli bacteria. The Scrambled aptamer did notrecognize any Listeria strains.

FIG. 16B shows that there is a synergistic effect between the twoaptamers, Ap03 and Ap08 when attached to the same beads, resulting in asensitivity of about 10e³ bacteria cells per system (10e⁴ PFU/ml)

These results also show the efficiency of using DMP to covalently bindthe aptamers to the BSA-DB, as well as its efficiently in blocking theprimary NH₂ groups on the BSA associated beads.

Example 9 Capture and Identification of Listeria Bacteria by TwoAptamers Assay

An assay, based on DB-BSA-aptamers and aptamer-SA-bDNA or aptamer-bDNA,was developed for the monitoring of the Listeria bacteria swab,re-suspended in PBS/BSA buffer.

Materials:

Aptamers were designed as described above (Ap03, Ap08, Scemb1).

Preparation of streptavidin-aptamers attached to ds-DNA (Ap03, Scemb1)and aptamers directly attached to ds-DNA (Ap03, Scemb1) were preformedas described above.

Dyna-Beads M280 Tosylactivated coated with BSA and covalently bound toNH₂-AP08 were preformed as described above, employing DMA for bothaptamers coupling and BSA NH₂ blocking.

Assay Construction:

DB-BSA-Ap08 preparations were washed, re-suspend in PBS/Tween-20 (0.01%)and aliquoted as needed. One colony of Listeria strain 4 (LS4) werecollected as a swab with a cotton stick and the bacteria wasre-suspended from the stick into 1000 ul of PBS/Tween/BSA.

To a pellet of 0.5 mg DB-BSA-AP08 complexes, 100 ul of bacteriapreparation were added and incubated for 1 h at RT by applying constantrotation. Samples were then washed five times with PBS-EDTA-Tween and100 μl of Aptamer-bDNA preparation were added and incubated for 60 minat RT.

Following three washings with PBS-Tween additional two washings withPBS-BSA-Tween, 100 μl of SA-HRP (1:5000) were added and incubated for 30min at RT. Samples were washed five times with PBS-BSA-Tween and 100 μlTMB were added and incubated for 10 min at RT. Reaction was stopped with100 ul Stop solution and samples were read at 450 nm within 30 min.

Results:

As shown in FIG. 17, the aptamer Ap03 recognized the Listeria bacteria(10e⁵ PFU), pre bound to the beads by the Ap08, while the scembl1aptamer did not recognize the beads bound bacteria. These results wereobtained with both aptamers-bDNA preparations, with or without SA,

Example 10 A Rapid Detection Assay for Penicillin-Binding Protein 2′(PBP2′)

Methicillin-resistant S. aureus bacteria have become a worldwide concernowing to their increasing frequency in hospitals, causing seriousstaphyloccal infections, including sepsis and endocarditis. Recentresearch suggests that in the identification of MRSA, it is moreaccurate to either directly detect the gene encoding the methicillinresistance determinant (mecA) or its product, penicillin-binding protein2′ and 2a (PBP2′ and PBP2a), which is found in the cell membrane of MRSA(Barrett D, et al Kinetic Characterization of the GlycosyltransferaseModule of Staphylococcus aureus PBP2. J. Bactrio. Mar. (2005) 2215-2217;Zeeshan M, et al. comparison of different phenotypic methods ofdetection of methicillin resistance in staphylococcus aureus with themolecular detection of mec-a gene. J. Coll. Physicians. Surg. Pak. 17(2007):666-70). As nucleic acid hybridizaion and DNA amplificationtechniques such as PCR and real time PCR for detecting the mecA gene areexpensive and technically demanding, simple and more inexpensivetechniques are required for routine use. The MRSA cell has about 2000PBP's per cell of which about 45% (900) are PBP2a and 25% (500) arePBP2′ (Dabelsteen E, Cell surface carbohydrates as prognostic markers inhuman carcinomas. The Journal of Pathology, 179: 358-369).

The detection assay of the invention is suitable to determineMethicillin-resistant S. aureus presence (MRSA) in a biological sampleby identifying the MRSA PBP2′/2a antigens. Rapid extraction of PBP2′from the bacterial membranes of MRSA is achieved by boiling the sampleunder alkaline conditions, followed by neutralization and centrifugationsteps (Farra A, et al. Role of outer membrane protein OprD andpenicillin-binding proteins in resistance of Pseudomonas aeruginosa toimipenem and meropenem. Int. J. Antimicrob. Agents. 31 (2008): 427-33.).This step, which destroys the bacteria, allows a safe workingenvironment during the remaining steps of the procedure. Using anti PBP2aptamers bound to magnetic beads, PBP2 is separated and concentratedflow the biological sample (e.g. whole blood, serum, plasma, and nasalor smear sample). This allows covalent binding of the addedSuccinimidyl-ds-bDNA to the PBP2. If required, an additional step ofamplification can be performed using the T7 RNA polymerase reaction(about 2 logs of bound ds-DNA) as described above. A positive PBP2′/2aantigen test result indicates the presence of MRSA and enables theselection of an appropriate drug treatment.

Example 11 Bacterial Sepsis Assay—Detection of Procalcitonin Levels

Procalcitonin (PCT), a species-specific propeptide of calcitonin, is aprotein of 116 amino acids with a molecular weight of 13 kDa. Underphysiological conditions, PCT is produced and then cleaved by a specificprotease to calcitonin and katacalcin in C-cells of the thyroid gland.However, strongly increased plasma concentrations of PCT were detectedin patients with thermal injury, in children with bacterial Meningitis,and in patients with sepsis and severe infection (Maruna et al.Physiology and Genetics of Procalcitonin Physiol. Res. 49 (2000): 57-61;Dondana et. al. Procalcitonin increase after endotoxin injection innormal subjects J. Clin. Endocrinol. Metab. 79 (1994) 1605-1608).

In comparison to that of sepsis patients (1000-100000 pg/ml), theconcentrations of PCT in the plasma of healthy blood donors were foundto be very low (around 40 pg/ml). Due to these characteristics and toits very long half-life in the blood (25-30 hours), PCT is routinelyused as a parameter for the diagnosis of severe bacterial and fungalinfections and for mediator-directed therapy of sepsis (Maruna et al.Physiology and Genetics of Procalcitonin Physiol. Res. 49 (2000): 57-61;Dondana et. al. Procalcitonin increase after endotoxin injection innormal subjects J. Clin. Endocrinol. Metab. 79 (1994) 1605-1608).

The detection assay of the invention is suitable to determine low andhigh levels of PCT in patient's body fluid sample.

Using anti PCT aptamers bound to magnetic beads, PCT is separated andconcentrated from whole blood, serum, plasma, nasal, smear, CSF oramniotic fluid sample. This allows the covalent binding of the addedSuccinimidyl-ds-bDNA to the PCT. If required, an additional step ofamplification can be performed using the T7 RNA polymerase reaction(about 2 logs of bound ds-DNA) as described above. The values obtainedfrom the assay are compared to a clinical reference table, driven fromestablished clinical data and are correlated with the severity ofbacterial sepsis.

Example 12 Detection of Cell Surface Associated Cancer Markers Using a“Two Aptamer” Assay

Tumor development is usually associated with changes in cell surfaceglycolipids and proteins. These changes include incomplete synthesis,over expression and modification (terminal carbohydrate structures) ofnormally existing cell surface substances, as well as the expression ofnew genes that are not usually expressed in that specific cell type.These changes which differentiate the cell from normal (non-malignant)cells and characterize it as a tumor cell are identified as “cellsurface cancer markers” (Olempska M, et al. Detection of tumor stem cellmarkers in pancreatic carcinoma cell lines. Hepatobiliary Pancreat DisInt. 6 (2007)).

Cancer cells can “break away”, “leak”, or “spill” from a primary tumor,enter lymphatic and blood vessels, circulate through the bloodstream,and settle down to grow within normal tissues elsewhere in thebody-metastasis. Most tumors and other neoplasms can metastasize,although in varying degrees. Therefore, tumor cells can be found in theblood circulation, as metastases as well as cancer cells associated withblood cells (Dabelsteen E, Cell surface carbohydrates as prognosticmarkers in human carcinomas. The Journal of Pathology, 179: 358-369).

The detection assay of the invention is suitable to determine even a fewtumor cells in the blood circulation, by the identification of specificcell surface markers (presence, i.e. in a qualitative assay or amount ina quantitative assay) on the surface of selected cells, without the needfor using a fluorescence microscope or FACS techniques. Using anti celltype directed aptamers bound to magnetic beads, the target cell isseparated and concentrated from whole blood samples. Adding a secondbiotinylated aptamer, which is directed against the selected marker,allows the binding of streptavidin ds-DNA. First amplification occurs asthere are many antigen marker copies per cell. Second amplification isoccurs during the T7 RNA polymerase reaction (about 2 logs of boundds-DNA). The values obtained from the assay are compared to a clinicalreference Table, driven from established clinical data, for thedetermination of tumor cell existence.

Example 13 Kits or Simultaneous Detection of Several Antigens

The kits of the invention may also be used for simultaneous detection ofseveral antigens in the same sample (for example a swab sample or a bodyfluid sample such as saliva, nasal secretion, blood, urine or feces).These simultaneous-multi-panel detection kits can be used in a “singleaptamer assay” or a “dual aptamer assay” as described below.

Multi-“single aptamer assay”: In stage-1 of the assay, different tubesare used each containing a different aptamer-beads complex, eachaptamer-beads complex is directed against a single selected target. Theremaining stages of the assay are performed as described above in thedescription of the “single aptamer assay”. The tube which generates afluorescence signal points to the presence of an antigen.

Some non-limiting examples include:

1) Hepatitis Kit: Suitable for the simultaneous detection of hepatitis Avirus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV) in apatient's serum sample. The importance of such a kit is based on theneeds to identify the virus in the case of a patient with hepatitissymptoms, as there is a different treatment for each virus.

2) Meningitis Kit: Suitable for the simultaneous detection ofEntero-virus, Herpes, CMV, Meningococcal, Pneumococcal, and Hib inpatients' CSF sample. The importance of such a kit is based on the needsto rapidly identify Meningitis as having a bacterial or a viral base, asthe severity of disease and mode of treatment differ according to thesource of the infection.

3) Respiratory Kit: Suitable for the simultaneous detection of SARS,Influenza A+B, Avian Influenza, Adeno virus and Rhino virus in nasalwash and swabs. The importance of such a kit is based on the needs torapidly identify the source of a respiratory infection, especially inthe case of pandemic threat, in hospitals and clinics, as well as in airand sea ports.

4) Blood coagulation kit: Suitable for the simultaneous detection of amissing/mutant blood coagulation factor in a patient's circulation. Theimportance of such a kit is based on the needs to identify the source ofa blood coagulation problem, to determine the appropriate treatment.

5) Tumor markers kit: Suitable for the simultaneous detection of solubletumor markers in the patient's circulation. The importance of such a kitis based on the need to identify the existence and progress of a tumorwithin a patient's body, for rapid and appropriate treatment.

Multi “Dual Aptamers Assays”

In stage-1 of the assay, different tubes are used each containing adifferent aptamer-beads complex, each aptamer-beads complex is directedagainst a single cell type selected marker. In stage-2 of the assay adifferent biotinylated aptamer, directed against a specific antigen onthe selected cells surface is added into each tube. The remaining stagesof the assay are performed as described above in the description of the“Two aptamer assay”. The tube which generates a fluorescence signalpoints to the presence of the cell type carrying the selected markerantigen.

Some non-limiting examples include:

1) Cell surface tumor markers kit: Suitable for the detection andidentification of metastases by the simultaneous recognition of cellstype and associated cell surface tumor markers, in a patient'scirculation. Among these are: CD133 on colon cancer cells, Est-1 onpancreatic and thyroid carcinoma cells, p63 on myoepithelial cells,EPM-1 and EXO-1 on gastrointestinal tumors cells, TEMs on tumorendothelial cells and CD44+CD24−/low on breast cancer cells. Theimportance of such a kit is based on the need to identify the existenceand progress of a tumor within a patient body, for rapid and appropriatetreatment.

2) Methicillin-resistant bacteria kit: Suitable for the detection andidentification of different methicillin-resistant bacteria strains,carrying the penicillin-binding protein 2 (PBP2), such as Staphylococcusaureus, pneumococci, Escherichia coli and Pseudomonas aeruginosa, and todistinguish between Methicillin-resistant S. aureus and borderlineoxacillin-resistant S. aureus (Balslev U et al. An outbreak ofborderline oxacillin-resistant Staphylococcus aureus (BORSA) in adermatological unit. Microb. Drug Resist. 11 (2005):78-81.). Accordingto this embodiment, the beads-associates aptamers are directed againsteach bacteria strain, and the biotinylated aptamer against a sharedepitop of the PBP2 antigens. The importance of such a kit is based onthe need to rapidly identify the drug resistant bacterial strains, asthere is a different life threatening situation and different treatmentfor each type of pathogen.

1-73. (canceled)
 74. A method for the detection of a target molecule in a sample comprising: a. obtaining at least one aptamer capable of binding to said target molecule, wherein said at least one aptamer is bound to a matrix; b. incubating said at least one aptamer which is bound to the matrix with the sample under conditions allowing the binding of the aptamer to the target molecule; thereby forming a matrix-aptamer-target molecule complex; c. contacting the matrix-aptamer-target molecule complex formed in step (b) with a polymer associated with a member of an affinity couple wherein said polymer further comprising a reactive group; thereby forming a matrix-aptamer-target molecule-polymer complex; and d. contacting said matrix-aptamer-target molecule-polymer complex with a complementary member of said member of an affinity couple, wherein said complementary member is associated with a detectable moiety, wherein the amount of said detectable moiety is indicative, of the presence of said target molecule in the sample, and wherein said target molecule is a viral antigen, a cancer marker or a soluble antigen selected from the group consisting of soluble cancer markers, inflammation-associated markers, hormones, cytokines, drugs, viral derived soluble molecules, bacterial derived soluble molecules and fungal derived soluble molecules.
 75. The method of claim 74 wherein the polymer is a nucleic acid molecule comprising a reactive group and further comprising a polymerase promoter sequence, thereby forming a matrix-aptamer-target molecule-nucleic acid molecule complex; and wherein the method further comprises a. adding a DNA or RNA polymerase enzyme and nucleotides associated with a member of an affinity couple under suitable conditions to affect DNA or RNA polymerization, thereby obtaining DNA or RNA molecules associated with a member of an affinity couple, and b. contacting said DNA or RNA molecules associated with a member of an affinity couple with a complementary member of said member of an affinity couple associated with a detectable moiety; wherein the amount of said detectable moiety is indicative of the presence of said target molecule in the sample.
 76. A method for the detection of a target molecule in a sample comprising: a. obtaining at least one first binding agent capable of binding to said target molecule, wherein said first binding agent is bound to a matrix; b. incubating said at least one first binding agent which is bound to the matrix with the sample under conditions allowing the binding of the binding agent to the target molecule; thereby forming a matrix-binding agent-target molecule complex; c. contacting the matrix-binding agent-target molecule complex formed in step (b) with a second binding agent-polymer complex, wherein said second binding agent-polymer complex is obtained by either i. obtaining at least one biotinylated second binding agent; ii. incubating said at least one biotinylated second binding agent with streptavidin thereby a biotinylated second binding agent streptavidin (b-binding agent-SA) complex is formed; and iii. incubating said b-binding agent-SA complex formed in step (ii) with a polymer associated with a member of an affinity couple wherein said polymer further having a reactive group thereby forming a second binding agent-polymer complex; or iv. obtaining at least one second binding agent, wherein said at least one second binding agent comprises a reactive group; and v. incubating said at least one second binding agent comprising a reactive group with a polymer associated with a member of an affinity couple wherein said polymer further having a reactive group thereby forming a second binding agent-polymer complex; d. contacting the matrix-binding agent-target molecule complex formed in step (b) with the second binding agent-polymer complex formed in step (c), under conditions allowing the binding of the second binding agent to the target molecule, thereby obtaining a target molecule-polymer complex; e. contacting said target molecule-polymer complex formed in step (d) with a complementary member of said member of an affinity couple associated with a detectable moiety, wherein the amount of said detectable moiety is indicative of the presence of said target molecule in the sample.
 77. The method of claim 76 wherein the second binding agent-polymer complex formed in step c is a second binding agent-nucleic acid complex, and wherein the polymer of step c (iii) or c (v) is a nucleic acid having an active group and further comprising a polymerase promoter sequence thereby forming a second binding agent-nucleic acid complex; and wherein the method further comprises a. adding a DNA or RNA polymerase enzyme and nucleotides associated with a member of an affinity couple under suitable conditions to affect DNA or RNA polymerization, thereby obtaining DNA or RNA molecules associated with a member of an affinity couple, and b. contacting said DNA or RNA molecules associated with a member of an affinity couple with a complementary member of said member of an affinity couple associated with a detectable moiety; wherein the amount of said detectable moiety is indicative of the presence of said target molecule in the sample.
 78. A method according to claim 74, wherein said aptamer comprises a reactive group at the 3′ and/or 5′ termini, wherein said reactive group is a member of an affinity couple selected from the group consisting of biotin/avidin, antigen/antibody, Molecular Imprinted Polymers/target ligand, protein-A/IgG, ligand/receptor, and a nucleic acid molecule/complementary sequence.
 79. A method according to claim 74, wherein said aptamer comprises reactive groups and said matrix is pre-coated with a protein, and wherein said aptamer is bound to the matrix by adding a cross linking agent, thereby forming an aptamer-matrix complex.
 80. A method according to claim 74, wherein said matrix is precoated with a molecularly imprinted polymer (MIP), an antibody, or a ligand.
 81. A method according to claim 80 wherein said aptamer-matrix complex is further incubated with a blocking agent, thereby blocking the remaining free active groups on the matrix and wherein said blocking agent is selected from a group consisting of DMP, citraconic anhydride, sulfo-NHS-Acetate, glutaraldehyde, photo-reactive groups, N-acetylcysteine and N,N′-Methanetetraylbis(2-propanamine) (DIPCDI) and 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC).
 82. A method according to claim 74 wherein said member of an affinity couple associated with the polymer is biotin.
 83. A method according to claim 74 wherein said polymer is a nucleic acid molecule and wherein said nucleic acid molecule comprises a reactive group at its 5′ and/or 3′ termini and wherein said reactive group is reactive with primary NH₂.
 84. A method according to claim 83 wherein said nucleic acid molecule is a single stranded nucleic acid forming a complex with biotin during a branched nucleic acid process or as a pre-prepared branch unit.
 85. A method according to claim 74 wherein said complementary member of said member of an affinity couple is a biotin-binding protein.
 86. A method according to claim 74 wherein said detectable moiety is an enzyme capable of catalyzing a reaction producing a detectable signal and a suitable substrate for said enzyme, wherein said enzyme is Alkaline Phosphatase (AP) or Horse Radish Peroxidase (HRP).
 87. A kit comprising: (a) a first binding agent; and (b) a second binding agent-polymer complex, wherein the polymer is associated with a member of an affinity couple and wherein said polymer is further associated with a reactive group.
 88. A kit according to claim 87 wherein said first binding agent is at least one aptamer; and wherein said second binding agent-polymer complex is a biotinylated polymer and wherein said biotinylated polymer is further associated with a reactive group.
 89. A kit according to claim 88 wherein said biotinylated polymer is a biotinylated nucleic acid molecule comprising a reactive group and further comprising a polymerase promoter sequence.
 90. A kit according to claim 87—wherein said reactive group is a succinimidyl ester group.
 91. A kit according to claim 87, further comprising at least one detection enzyme and optionally a substrate for said detection enzyme.
 92. A method for the diagnosis of a pathological condition in a subject comprising using a detection method in accordance with claim 74, wherein said target molecule is a target molecule associated with the pathological condition and wherein the amount of said detectable moiety is indicative of the presence of a pathological condition in the subject, and wherein said pathological condition is cancer, an autoimmune disease, or a viral, bacterial or fungal infection.
 93. A method for monitoring the efficiency of a therapeutic regimen in a subject suffering from a pathological condition comprising using a detection method in accordance with claim 74, wherein said target molecule is an antigen associated with the pathological condition and wherein the amount of said detectable moiety is indicative of the level of the pathological condition and thereby of the efficiency of the therapeutic regimen in the subject, and wherein said pathological condition is cancer, an autoimmune disease, or a viral, bacterial or fungal infection. 